Name: system for efficacy and cytotoxicity screening of inhibitors targeting intracellular mycobacterium tuberculosis
Uploaded: 2017-04-05T14:30:00.000+00:00
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Description: Watch this Scientific Journal Video about System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis at JoVE.com

This work was supported by BC Lung Association and Mitacs.

1. Estirpe bacteriana e Meio de Crescimento <ol><li> Adicione de dextrose e albumina de solução de estoque de sal (ADS) por solubilização de 25 g de albumina de soro bovino, 10,0 g de dextrose, e 4,05 g de cloreto de sódio em 460 mL de água desionizada. Filtro-esterilizar a ADS e armazenar a 4 ° C. </li><li> Adicione 7H9 pela adição de 4,7 g de 7H9 em pó e 2 ml de glicerol a 900 mL de água purificada. Autoclave o 7H9 a 121 ° C durante 10 min e permita que arrefeça para a temperatura ambiente antes de continuar. Adicione 7H9ADST por adição de 100 mL de ADS e 0,5 ml de Tween 80 a 900 mL de caldo 7H9. Armazenar a 4 ° C. </li><li> Pesar 50 mg de sulfato de canamicina e dissolver em 1 ml de água desionizada; a concentração final é de 50 mg / mL. Filtro-esterilizar e armazenar a -20 ° C. Adicionar 0,5 mL de canamicina solução estoque por 1 L de 7H9ADST. NOTA: Este meio deve ser feita, de modo dimensionar os volumes apropriadamente de acordo com o tamanho da cultura. </li><li> Crescer M. tuberculosis em 7H9ADST suplementado com canamicina em pé cultura. Agite a cultura diariamente e dilui-lo antes da DO600 atinge 1,0 para evitar aglomeração. NOTA: A estirpe de M. tuberculosis utilizado para o desenvolvimento deste método foi H37Rv transformada com o plasmídeo pJAK2.A 12. pJAK2.A é um plasmídeo integrativo baseado no vector pMV361, que permite a expressão de alto nível do gene luciferase de pirilampo do promotor hsp60 e pode ser seleccionado utilizando canamicina. </li></ol> 2. THP-1 e Meio de Manutenção <ol><li> Adicionar 50 mL de soro inactivado por calor de bovino fetal (FBS) e 5 mL de 200 mM de L-glutamina e 500 mL de meio RPMI 1640 para fazer meio RPMI incompleta (cerca de 10% de FBS e glutamina 2 mM). </li><li> Manter uma cultura de células THP-1 de acordo com o protocolo padrão 13. Resumidamente, crescer as células THP-1 em meio RPMI incompleta mantendo ao mesmo tempo uma densidade celular de 0,2 a 1 milhão por ml de meio entre passábios. </li></ol> 3. Apresentação de intracelular de alto rendimento usando-expressar luciferase M. tuberculosis H37Rv <ol><li> Medir a densidade óptica de uma suspensão de bactérias em crescimento activo num espectrofotómetro a um comprimento de onda de 600 nm. Calcular a densidade bacteriana utilizando o factor de conversão de 0,1 OD 600 = 3 x 10 7 de bactérias por ml. </li><li> Pipetar as bactérias suficientes para uma multiplicidade de infecção (MOI) de 10: 1 para um novo tubo de centrífuga. Sedimentar a 3000 xg durante 10 min e aspirar o líquido. Adicionar 50 ul de soro humano a 450 uL de RPMI 1640. Dimensionar o volume de se apropriar valores para a experiência. </li><li> Para opsonizar as bactérias, ressuspender o sedimento a uma densidade de 1 x 10 8 de bactérias por 500 ul de RPMI1640 contendo 10% de soro humano. Deixar a mistura a incubar a 37 ° C durante 30 min. Determinar a densidade da cultura de células THP-1 por contagem com um hemocitómetro e um microsco invertidoPE. </li><li> Sedimentar as células em tubos de centrífuga estéreis, a 100 x g e 37 ° C durante 10 min. Aspirar o sobrenadante e ressuspender as células em meio RPMI incompleta a uma densidade de 1 milh de culas por mL. Adicionar forbol-12-miristato-13-acetato (PMA) para uma concentração final de 40 ng / mL. NOTA: Este será referido como o mix diferenciação. </li><li> Combinar opsonizado de M. tuberculosis com THP-1 mistura de diferenciação, a uma MOI de 10: 1 e alíquotas da mistura final em 100 ul por cavidade em uma placa branca de 96 pos de fundo plano. Regularmente agita-se a mistura para assegurar a uniformidade. Permitir que a diferenciação e a infecção prosseguir durante a noite a 37 ° C numa incubadora humidificada contendo 5% de CO 2. </li><li> Lavam-se os poços duas vezes com 100 uL de meio RPMI cada. Adicionar compostos diluídos para as concentrações desejadas em RPMI incompleta e incubar durante 3 dias. </li><li> Aspirar o meio das cavidades. Adicionar 50 uL de reagente de ensaio de luciferase para cada cavidade. Selar as placas com transparent selantes de placas adesivas. Permitir 5 min de incubação a 22 ° C e, em seguida, obter uma leitura num luminómetro em 1 s por poço. </li></ol> 4. Análise de Citotoxicidade Usando um grupo 3- (4,5-dimetiltiazol-2-il) -2,5-difeniltetrazólio (MTT) 14 <ol><li> Diferenciar células THP-1 em meio RPMI incompleto suplementado com 40 ng / ml de PMA, em placas de 96 poços claras. Manter uma densidade celular de 1 milhão por ml e alíquota de 100 uL por poço. Permitir diferenciação prosseguir durante a noite a 37 ° C numa incubadora humidificada contendo 5% de CO 2. </li><li> Aspirar o meio dos poços e lava-se duas vezes com RPMI 1640. Adicionar compostos diluídos em RPMI incompletos para os poços. Incuba-se durante 3 dias. </li><li> Dissolve-se 0,5 g de MTT em 100 ml de solução salina tamponada com fosfato (PBS) para produzir uma solução stock de 5 mg / mL. Filtrar em condições estéreis e armazenar a -20 ° C, ao abrigo da luz; é melhor fazer esta solução fresca. </li><li> 2,5 horas antes da suae o fim do período de incubação de 3 dias, adicionar 25 ul de solução de MTT a cada poço e completar o período de incubação. </li><li> Prepare a 50% de N, N-dimetil formamida (DMF) por mistura de 250 mL de DMF com 250 mL de água desionizada. </li><li> Preparar tampão de extracção de MTT como se segue: Pesar 100 g de SDS em um frasco de 500 ml e adicionam-se 300 mL de 50% de DMF. Aplicar fogo baixo para permitir que o SDS para dissolver. Adicionam-se 10 mL de ácido acético puro e 12,5 mL de HCl 1M. Encha até à marca de 500 mL com 50% de DMF; a composição final do tampão de extracção é de 50% de DMF, 20% de SDS, ácido acético a 2,5%, e 2,5% de ácido clorídrico 1 M. </li><li> No final do período de tratamento, adiciona-se 100 uL de tampão de extracção (aquecida a 45 ° C para dissolver todos os cristais) para cada poço. Deixar a mistura a incubar durante a noite a 37 ° C numa incubadora humidificada contendo 5% de CO 2. Leia a absorvância a 570 nm. NOTA: O ensaio de citotoxicidade é melhor realizado em paralelo com um intracelulartela ular usando mesmo lote e idade das células THP-1. </li></ol> 5. No caldo Análise Atividade Usando um resazurina Ensaio 3 <ol><li> Crescer M. tuberculosis em 7H9ADST à fase mid-log (~ 0,5-0,8 OD 600). Dilui-se a cultura com a 7H9ADST a 0,01 OD600. Dilui-se os compostos em 7H9ADST 2x as concentrações de teste e alíquota de 100 ul de cada composto diluída para cada poço. </li><li> Transferir 100 ul da suspensão bacteriana diluída para cada poço. Permitir que as placas a incubar a 37 ° C num incubador humidif içado, durante 5 dias. Dissolve-se 10 mg de resazurina em 100 ml de água desionizada e o filtro esterilizado. </li><li> Adicionar 30 mL de solução de resazurina e controlar a mudança de cor após 48 h; o crescimento bacteriano é indicado por uma conversão de cor de azul para rosa. NOTA: A análise quantitativa também pode ser realizada medindo quer a fluorescência a 590 nm com excitação a 530-560nm ou a absorvência a 570 nm e 600 nm de 15. </li></ol>

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The authors declare no competing financial interests for this work.

O objetivo deste estudo foi o de criar um método HTS simples e de baixo custo utilizando um modelo de infecção intracelular humano para o M. tuberculosis. A tuberculose é uma doença humana caracterizado pela infecção de macrófagos alveolares por M. tuberculosis. Devido a questões de biossegurança, pesquisa envolvendo modelos biológicos, tanto da bactéria e as células hospedeiras tem sido usado no passado. No entanto, foi demonstrado que o uso de bactérias substitutos e modelos não-humano...

Mycobacterium tuberculosis, o agente causador da tuberculose (TB), é uma das principais causas de morbilidade e mortalidade em todo o mundo. TB sensível a drogas é uma doença tratável que requer múltiplos antibióticos durante um período de 6 meses. Apesar de ser uma doença tratável da taxa de mortalidade foi estimada em 1,5 milhões em 2015 um. Nos últimos 10 anos, tem vindo a aumentar as preocupações com a prevalência de fármaco-resistentes de M. tuberculosis. Multirresistente tuberculose (TB-MR) é definida como a tuberculose que é resistente a pelo menos isoniazida (INH) e Rifampicina (RMP), e a maioria das estirpes de MDR-TB são também resistentes para seleccionar fármacos para a tuberculose de segunda linha, o que limita as opções de tratamento . Os efeitos da resistência à droga criar um maior desafio para o tratamento de pacientes co-infectados com o Vírus da Imunodeficiência Humana (HIV); 400.000 doentes em todo o mundo morreu de tuberculose associada ao HIV em 2015 um. Lamentavelmente, os Estados Unidos Food and Drug Administração aprovou apenas uma nova droga TB contra a MDR-TB, bedaquilina, nos últimos 40 anos 2. Avanços em encontrar melhores e mais curtas terapias de tuberculose são urgentemente necessários, a fim de permanecer à frente na luta contra a TB e MDR-TB. Tradicionalmente, as telas de drogas de TB são realizadas sob condições de crescimento in vitro em meio de crescimento, através do qual os compostos são adicionados a uma cultura em crescimento e a sua eficácia na erradicação dos microrganismos são determinadas pela contagem de unidades formadoras de colónias (CFU) em meio sólido. Como contagens de UFC é trabalho intensivo, demorado, e caro, vários métodos indiretos foram desenvolvidos para aliviar este problema. Tais métodos incluem o ensaio de viabilidade Alamar Blue 3, a determinação da fluorescência a partir de 4 proteína verde fluorescente (GFP) ou luminescência 5 a partir de bactérias que expressam luciferase, e a estimativa do trifosfato de adenosina total de (ATP) 6, 7. TB típica é caracterizada por uma infecção de M. tuberculosis do pulmão, onde residem as bactérias e replicar no interior das fagossomas de macrófagos alveolares 8. A tela fenotípica simples no caldo pode atender patógenos extracelulares; no entanto, na perspectiva histórica, bateu compostos contra M. tuberculosis identificados usando este método muitas vezes não conseguem corresponder às expectativas durante as etapas de validação de transformação em modelos de infecção. Propomos que a droga TB é melhor executada em um modelo de infecção da célula hospedeira intracelular. No entanto, os modelos intracelulares possuem muitos obstáculos tecnológicos e biológicos para triagem (HTS) desenvolvimento de alto rendimento. Um grande obstáculo é a complexidade do processo de infecção, exemplificado por numerosos passos e a remoção de bactérias extracelulares elaborada por no meio de lavagem. Um segundo grande obstáculo é o longo tempo de requirements, como a detecção de crescimento, normalmente feito por contagem de CFU nas placas de cultura, é um processo que demora mais de 3 semanas para completar. Uma solução para substituir contagens de UFC foi fornecida por microscopia fluorescente automatizada em combinação com bactérias fluorescentes. No entanto, esta solução requer um investimento inicial equipamento que está fora do alcance de muitos laboratórios de pesquisa. Uma simples, de baixo custo, e método HTS doença relevante aumentaria grandemente o processo de descoberta de drogas. Neste estudo, são descritos um novo sistema de HTS, modular que visa proporcionar uma rápida e altamente flexível, mas económica, ensaio adequado para a determinação da actividade de compostos contra intracelular de M. tuberculosis. Este sistema é composto por três módulos: (i) intracelular, (ii) da citotoxicidade, e (iii) Ensaios in-caldo. O resultado final combinada fornece uma descrição detalhada das propriedades do composto, com a informação adicional quanto ao modo de potencial de acção. este screening sistema tem sido utilizado em vários projectos com várias bibliotecas de compostos, o modo de acção que têm como alvo, incluindo a análise de sinergia droga 9, a estimulação de autofagia 10, e a inibição de M. tuberculosis -secreted factor de virulcia (não publicado). Os compostos de modo desconhecido de ação também foram estudados 11. Uma versão modificada deste método foi também adoptada pelo nosso parceiro industrial como o método de rastreio primário de identificar novos compostos contra intracelular de M. tuberculosis 11.

<table><tbody><tr><td>RPMI 1640</td><td>Sigma-Aldrich</td><td>R5886</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific</td><td>12483020</td><td></td></tr><tr><td>Middlebrook 7H9</td><td>Becton, Dickinson and Company</td><td>271210</td><td></td></tr><tr><td>Tween80</td><td>Fisher Scientific</td><td>T164</td><td></td></tr><tr><td>Albumin, Bovine pH7</td><td>Affymetrix</td><td>10857</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>BP350</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358</td><td></td></tr><tr><td>kanamycin sulfate</td><td>Fisher Scientific</td><td>BP906</td><td></td></tr><tr><td>PMA</td><td>Sigma-Aldrich</td><td>P8139</td><td></td></tr><tr><td>MTT</td><td>Sigma-Aldrich</td><td>M2128</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide (DMF)</td><td>Fisher Scientific</td><td>D131</td><td></td></tr><tr><td>1 M Hydrocholoric acid (HCl)</td><td>Fisher Scientific</td><td>351279212</td><td></td></tr><tr><td>Acetic acid</td><td>Fisher Scientific</td><td>351269</td><td></td></tr><tr><td>SDS</td><td>Fisher Scientific</td><td>BP166</td><td></td></tr><tr><td>Resazurin</td><td>Alfa Aesar</td><td>B21187</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229</td><td></td></tr><tr><td>THP-1</td><td>American Type Culture Collection</td><td>TIB-202</td><td></td></tr><tr><td>&lt;em&gt;M. tuberculosis&lt;/em&gt; H37Rv</td><td></td><td></td><td></td></tr><tr><td>96-well flat bottom white plate</td><td>Corning</td><td>3917</td><td></td></tr><tr><td>95-well flat bottom clear plate</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>Transparent plate sealer</td><td>Thermo Fisher Scientific</td><td>AB-0580</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>Biomate 3</td><td></td></tr><tr><td>Microplate spectrophotometer</td><td>Biotek</td><td>Epoch</td><td></td></tr><tr><td>luminometer</td><td>Applied Biosystems</td><td>Tropix TR717</td><td></td></tr></tbody></table>

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.

Rastreio intracelular de alto rendimento usando M. tuberculosis expressando o gene da luciferase A Figura 2A e na Tabela 1 contém os dados brutos recolhidos pelo luminómetro, expressa em unidades luminescentes relativas (RLU), mostrando o efeito de uma concentração crescente da droga rifampicina em M. tuberculosis TB no interior das células THP-1. A Figura 2A</stron...

Watch this Scientific Journal Video about System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis at JoVE.com

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

Nós desenvolvemos um sistema de rastreio de alto rendimento modular para descobrir novos compostos contra o Mycobacterium tuberculosis, direccionamento intracelular e em caldo de crescimento de bactérias, bem como a citotoxicidade contra a célula hospedeira de mamífero.

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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

8.5K visualizações.  University of British Columbia.  Nós desenvolvemos um sistema de rastreio de alto rendimento modular para descobrir novos compostos contra o Mycobacterium tuberculosis, direccionamento intracelular e em caldo de crescimento de bactérias, bem como a citotoxicidade contra a célula hospedeira de mamífero. 

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

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) ...

Imunologia e Infecção

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Um ensaio fenotípico microscópico para quantificação de micobactérias intracelulares adaptadas para triagem de alto rendimento/alto conteúdo

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

Visualização do carvão Agar resazurina Ensaio para a semi-quantitativa, médio rendimento enumeração de micobactérias

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

High Throughput, em tempo real, Teste Dual-leitura dos intracelular Actividade Antimicrobiana e citotoxicidade celular eucariótica

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

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

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

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

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

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 ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

Um Sistema de Cultura Automatizado para Uso em Testes Pré-Clínicos de Terapias Dirigidas ao Hospedeiro para Tuberculose

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

<meta charset="utf8"></head><body>Ensaio de triagem de alto rendimento para Mycobacterium abscessus usando repórteres duplos

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

<meta charset="utf8"></head><body>Autor em destaque: Protocolo de triagem de drogas escalável para descoberta eficiente de tratamentos com M. abscessus

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Desenvolvimento de ensaio para triagem de medicamentos de alto rendimento contra micobactérias

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Acelerando a determinação da tuberculose usando o método de micro-UFC

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

Autor em destaque: Otimizando a determinação de UFC para avaliação eficiente da eficácia da vacina contra TB e análise de apresentação de antígeno 

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

Ensaio de Unidade Formadora de Microcolônias para Avaliação da Eficácia de Vacinas Contra Tuberculose

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

Sistema de Eficácia e citotoxicidade Rastreio de inibidores Segmentação intracelular

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