Name: a high-throughput compatible assay to evaluate drug efficacy against macrophage passaged mycobacterium tuberculosis
Uploaded: 2017-03-24T15:00:00.000+00:00
Duration: 10 min 29 s
Description: Watch this Scientific Journal Video about A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis at JoVE.com

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.

注：2 6206は、非病原性株17、18である結核菌の MCとしては、このプロトコルですべての作業は、バイオセーフティレベル2の施設（BSL-2）で行うことができます。 結核菌 MC 2を発現する緑色蛍光タンパク質1.培養条件6206（ マウンテンバイク -GFP） 注： ヒト結核菌H37RvはpMN437がこのプロトコル16全体で使用されるプラスミドを発現するGFPで形質転換された栄養要求株MC 2 6206（ΔpanCD、ΔleuCD）に由来しました。研究者のための歪みを簡単にアクセシビリティを有効にするには、野生型株を発現する非GFPでのMtb -GFP株を代用することが可能です。しかし、GFP発現は、食作用とのMtb /マクロファージの凝集体の形成の視覚的確認を可能にすることが望ましいです。経度のためのGの長期保存、Mtbの -GFPを、20％グリセロールを補充し（ステップ1.1参照）完全7H9培地中で-80℃で凍結しました。 <ol><li> 10％ミドルOADC、0.02％チロキサポール、24μgの/ mLのD-パントテン酸、を50μg/ mLのL-ロイシンおよび50μg/ mLのハイグロマイシンBで7H9培地を補充することによって、完全なマウンテンバイク -GFPメディア（7H9-C）を準備</li><li> マウンテンバイク -GFPの1つのバイアルを解凍し、30 mLの正方形の底PETGフラスコ中の7H9-Cの10 mLに追加します。 </li><li>ロータリーシェーカー（50回転）に37℃でインキュベートします。 OD 600が 1.0に達するまで（約5-8日）数日おきに光学密度測定（OD 600）を取ります。 </li><li> 0.2〜1.0の間のOD 600を維持するために必要に応じて希釈して継代培養。 </li><li>感染のために、対数増殖期（OD 0.6から1の600）内のMtb -GFPの確立された文化を使用します。塊状の文化を避けるために、水浴中のMtb培養フラスコを超音波処理（130 W; 3×5秒パルス）ONCeまたは週二回</li></ol> 2.培養条件 THP-1細胞<ol><li> 10％熱不活性化ウシ胎児血清、2mMのL-グルタミン、および10mM HEPESを含むRPMI 1640培地を補充することによって、完全なTHP-1細胞培地（RPMI-C）を調製します。 </li><li> 5％CO 2の加湿雰囲気中37℃でT-75フラスコ中で細胞をインキュベートします。 </li><li>血球計数器を用いて細胞数をカウントしたり、一日おきにフローサイトメトリーおよび0.1 mLの100万〜0.6の間の濃度でTHP-1細胞を維持します。 </li></ol> 3.感染プロトコルのMtb /マクロファージ集計構造を生成するために、 <ol><li> THP-1細胞調製物（96ウェルプレートあたり） <ol><li> 5分間250×gで遠心分離し、7×10 6 THP-1細胞。 7 mLのRPMI-Cでの再懸濁。 </li></ol></li><li> MTB -GFP準備 <ol><li>遠心分離2.8×10 8 のMtb -GFPスイングバケット遠心分離機で3200×gで5分間のuOD 600 1.0 = 3×10 8個の細菌/ mLでの変換を歌います。 </li><li>ステップ3.2.1のようにRPMI-Cと遠心機で一回洗浄します。 </li><li> 10秒のための7 mLのRPMI-Cで再懸濁し、ボルテックス。 </li></ol></li><li> 感染 注：テンプレートについては、 図1を参照してください。 <ol><li>培地の蒸発を防ぐために、水のリムの行AとHと、列1および12に滅菌水200μLを加えることにより、96ウェルプレートを準備します。 </li><li>バックグラウンドコントロール（ブランク）用カラム2（G2へのB2）に200μLのRPMI-Cを追加します。 </li><li>感染させるために、THP-1細胞懸濁液（ステップ3.1）へのMtb -GFP懸濁液（ステップ3.2）を追加し、ピペットでよく混ぜます。最終的なTHP-1細胞密度は、mL当たり5×10 5であり、感染の対応する多重度は40です。 </li><li> 25 mLのリザーバにTHP-1 / マウンテンバイク -GFP懸濁液を注ぎます。 </li><li> usin残りのすべての96ウェル（G11を通じてB3）にTHP-1 / マウンテンバイク -GFP懸濁液200μLを追加GAマルチチャンネルピペット。 注：複数の96ウェルプレートで作業する場合は、定期的にさえ混合物を各ウェルに添加されていることを確認するために、リザーバ内の残りのTHP-1 / MTBサスペンションを再懸濁します。 </li><li> 7〜10日間、5％CO 2で37℃でインキュベートします。 </li><li>ゆっくりと100μLを除去することにより、2日おきにメディアを変更し、マルチチャンネルピペットを用いて100μLに予め温めたRPMI-Cを追加するだけでなく、静かにそれぞれの最上部からメディアを過ごしました。井戸を再懸濁し、ウェルの底にマウンテンバイク /マクロファージの凝集体を邪魔しないでください。 </li><li>視覚のMtb /マクロファージの凝集体の大きさに着目し、毎日の蛍光顕微鏡（4-10X目的）によってウェルを調べます。日7-10では、 マウンテンバイク /マクロファージの凝集体は、薬物の効力試験（第4節）のために進行する（参考のために図2を参照）を十分に大きくなければなりません。 </li><li>所望であれば、自動細胞イメージングシステムを用いて96ウェルのキャプチャー画像が嵌め込ま明視野およびGFPフィルターでマウンテンバイク /マクロファージ凝集体形成を文書化するために設定します。 注：ユーザーが自動化されたイメージングシステムへのアクセスを持っていない場合は、代表ウェルの画像をGFPと明視野能力を有する任意の適切な顕微鏡を用いて撮影することができます。 </li></ol></li></ol> マウンテンバイク /マクロファージ集約由来医薬品に対する効力のMtbを評価する4.成長阻害アッセイ<ol><li> 薬剤の準備（2薬剤のための三重の条件） 注：テンプレートについては、 図1Aを参照してください。 <ol><li>独立した96ウェルプレートでは、G10までB2に7H9-Cメディアの125μLを追加します。 </li><li>ステップ4.2.4での希釈を考慮するために、7H9-Cの1mLに二重最高所望の最終濃度で、二つの薬剤を準備します。 </li><li>それぞれ三連の治療のために、ウェルB11-C11-D11とE11-F11-G11に各薬剤の250μLを追加します。 </li><li>マルチチャンネルピペットを使用して、連続希UTE試験薬物B10-G10にB11-G11から125μLを移動することで2倍。各ステップで5回ピペッティングすることによって混合します。 </li><li>プレートを横切って（右から左）の列に列から125μLを移動させ、カラム4の後に停止し続けます。 </li><li>カラム4を混合した後、廃棄物容器に125μLを捨てます。列2と3は、バックグラウンド（ブランク）とプラス成長コントロールなどの使用を可能にするために、任意の薬を含めることはできません。 注：また、テスト薬剤のライブラリについては、図1（b）のテンプレートに従ってください。各96ウェルプレートは、単一の濃度で58の薬物を収容することができます。また、ステップ4.2.4で半分に希釈されるので、倍の所望の最終濃度を使用して、この薬物プレートを準備します。 </li></ol></li><li> 薬物治療： <ol><li> Mtbを含む96ウェルプレートを取り出し、マクロファージ（ のMtb /マクロファージ凝集体）-infected。 </li><li>慎重にマルチチャンネルピペットを用いて、関連するすべてのウェル（G11を介してB2）をデカント。2段階の方法でこれを実行します。第一プレートを傾けることなく、150μLを削除してから、プレートを傾けるとウェルの底縁にピペットチップを挿入することにより、残りのメディア（〜50μL）を取り外します。 マウンテンバイク /マクロファージの凝集体がウェルの底に付着しているように、何の材料が失われてはなりません。 </li><li>静かマウンテンバイク /マクロファージの凝集体を含むプレートに関連するすべてのウェル（G11を通じてB2）に7H9-Cメディアの100μLを追加します。 </li><li>マルチチャンネルピペットを用いて、感染プレートの対応するウェルに96ウェルプレート（ステップ4.1）を含む薬剤から100μLを移します。 </li><li>密封された袋に入れて、37℃で3日間インキュベートします。 </li></ol></li></ol>レサズリンマイクロタイターアッセイを使用して5薬効の定量<ol><li> H 2 O中の0.8mg / mLの最終濃度でレサズリン原液を準備し滅菌のため0.22μmの細孔径のPVDF膜を通して濾過。</li><li> 1：1の比率で2レサズリンストック溶液、H 2 OおよびTween-80（H 2 O中20％溶液）を混合して溶液を作動レサズリン準備します。最終濃度は0.4 mg / mlでのレサズリン及び5％のTween-80です。 </li><li> 37℃で24時間、530 nm励起および590 nmの発光で30分毎に蛍光を読み取るためのプログラムは、プレートリーダーを使用してセットアップ。 37°Cにプリ暖かいプレートリーダー。 </li><li>マルチチャンネルピペットを用いて薬剤処理プレート（ステップ4.11）に関連するすべてのウェル（G11を通じてB2）にレサズリン作業溶液20μLを追加します。 </li><li>プレートリーダーでプレートを置き、ステップ5.3で設定プログラムを起動します。 注：アッセイプレートの大量の処理を容易にするために、37℃のインキュベーター中でアッセイを開発し、単一の蛍光は24時間毎に読み出し実行するのに十分です。これはまた、運動やインキュベーション機能を備えたプレートリーダーへのアクセス権を持たないユーザーにも適用可能です。 </li></ol>

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The authors have nothing to disclose.

ここでは、詳細に薬効試験に適した代替のMtb感染モデルを説明してきました。このモデルは、アカウント初期の結核治療薬の開発プロセスの間に、より配慮を与えられるべき二つの重要な要因を考慮に入れる：薬剤浸透と感染時のMtbの代謝変化に生理学的に関連の障壁の存在。我々は以前に私たちの感染モデルの利点を示し、スループットの互換性16の?...

<html>結核（TB）は、40年以上にわたって1用の抗結核化学療法レジメンの利用可能性にもかかわらず、世界的に深刻な健康への脅威のまま。これは、非準拠2を患者につながる複数の薬剤の組み合わせを使用して6ヶ月以上の長い治療期間の要件に一部起因します。近年の薬剤耐性結核の出現は、さらに臨床的に承認された医薬品の開発に成功が事実上存在しない3分野で問題を配合しました。確かに、徹底的な抗結核薬の開発にもかかわらず、唯一の単一の薬物は、FDAは、過去40年4臨床使用が承認されました。したがって、抗結核薬の新しい世代が緊急にこの問題に対処するために必要とされます。 結核治療薬の発見において重要な問題は、臨床設定における有効性へのin vitroでの活性を有する化合物からの転送成功の欠如であります= &quot;外部参照&quot;&gt; 5、6、7。最初に、ターゲットベースのアプローチは、細菌細胞全体に変換することができなかった抗Mtbの薬剤 5をスクリーニングするために使用されました。 Mtbの細胞を用いた場合でも、それは多くの場合正確にインビボ 8,9 において薬効を予測していないブロス増殖させた培養物を用いて行われます。これらの問題は認識されているとのMtbを含むマクロファージまたは潜在性のMtbに対する薬物スクリーニングアッセイは、正常8に確立された、10、11、12。しかし、これらのより高度なアッセイは、薬は、非血管新生した肺病変における、および感染部位に壊死巣で遭遇する浸透障壁に十分な配慮を与えることはありません。確かにでも最初の行の結核治療薬のリファンピシンのため、次善の投与は、 生体組織や脳脊髄液（CSF） で不十分なに疑問視されている13、14、15だけでなく、細胞内のMtb 8、9に対して有効性を減少を浸透。このように、初期のリード開発プロセスの間に考慮に入れ、これらのパラメータを取る新モデルおよびアッセイは間違いなく結核治療薬の発見の努力を改善するであろう。 このニーズに応えるために、我々は最近、安価で迅速、かつMtbの薬効試験の16のためのBSL-2互換の代替感染モデルを確立しました。生理学的に関連する細胞透過障壁を再現し大マクロファージの結合構造体、および生成されたマクロファージ継代以内に密集したMtbの生産この感染モデル<e潜在マウンテンバイクに似ている、変化した生理状態を持つメートル&gt;マウンテンバイク。この感染モデルから派生したMtbは、他の細胞内感染モデルと一貫性のある結果を生成した薬効を評価するためにレサズリンマイクロタイターアッセイ（REMA）と合わせ、血清濃度に比べて高いCSF濃度を達成するための一般的な結核治療薬の報告能力とよく相関していました16。 ここでは、 マウンテンバイク /マクロファージの結合構造体の生成は、REMAを使用して、薬剤感受性試験に適したマクロファージ継代のMtbを生成するために、詳細に説明します。特に、我々は、この感染系を候補の抗結核薬のスループットスクリーニングとの互換性のために96ウェル形式に適合させることができる方法を示します。

<table><tbody><tr><td>7H9</td><td>BD Difco</td><td>271310</td><td>Follow manufacturer&#039;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 H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>D-Pantothenic acid hemicalcium salt</td><td>Sigma</td><td>P5710</td><td>Prepare 24 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>L-leucine</td><td>MP Biomedicals</td><td>194694</td><td>Prepare 50 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>Hygromycin B</td><td>EMD Millipore</td><td>400051</td><td>Prepare 200 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O</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&amp;nbsp;</td><td>Fisher Scientific</td><td>BP2679250</td><td>Prepare 10 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Moxifloxacin Hydrochloride</td><td>Acros Organics</td><td>457960010</td><td>Prepare 10 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Resazurin Sodium Salt</td><td>Sigma</td><td>R7017</td><td>Prepare 800 &amp;mu;g/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>Tween-80</td><td>Fisher Scientific</td><td>T164500</td><td>Prepare 20% stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; 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.

96ウェルプレートフォーマットにこの感染モデルを適合させるの頑健性を確認するために、ここでは、図に示したテンプレートに従ってリファンピシン（RIF）及びモキシフロキサシン（MOXI）に当社の96ウェル適応感染モデル由来のMtbの薬剤感受性を調べました1A。我々は、このアッセイの鍵のMtb /マクロファージ集合構造の発生?...

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.

マクロファージ継代に対する薬効を評価するために、ハイスループットアッセイの互換性<em&gt;結核菌</em

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

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Research

JoVE Journal

Immunology and Infection

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

7.7K ビュー.  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. 

ビデオ: 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.

の成長結核バイオフィルム

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

免疫・感染学

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.

高スループット/高含有スクリーニングに適応した細胞内マイコバクテリアの定量化のための顕微鏡表現型アッセイ

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.

マイコバクテリアの半定量的、ミディアムスループット列挙のためのチャコール寒天レサズリンアッセイの可視化

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

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.

細胞内の標的阻害剤の有効性及び細胞毒性スクリーニングのためのシステム<em&gt;結核菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

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.

結核の宿主向け治療の前臨床試験に使用する自動培養システム

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>デュアルレポーターを用いた Mycobacterium abscessus のハイスループットスクリーニングアッセイ

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>著者スポットライト: M. abscessus 治療の効率的な発見のためのスケーラブルな薬物スクリーニングプロトコル

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.

マイコバクテリアに対するハイスループット薬物スクリーニングのためのアッセイ開発

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

Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1

The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic strains and genetically engineered cells. Here, an alternative method to detect biochemical interaction between the macrophage microbial sensor SLAMF1 and Mycobacterium tuberculosis is described. Two technical approaches employing flow cytometry and fluorescence microscopy were developed. Total cell protein extracts from human macrophages were generated, then incubated with whole cells of M. tuberculosis (WCMtb)&#160;or M. tuberculosis antigens (Mtb Ags) overnight at 4 &#176;C and finally cross-linked using formaldehyde/glycine/ethylene glycol bis (succinimidyl succinate) treatment. SLAMF1 interaction with WCMtb by flow cytometry was detected with a PE-specific anti-SLAMF1 antibody. The existence of interaction by fluorescence microscopy was performed by attaching Rhodamine-PE stained Mtb Ags to poly-D-lysine coated slides, which were incubated with the total protein extract from monocyte-derived macrophages. After cross-linking treatment, SLAMF1 was visualized using primary (anti-SLAMF1) and secondary (Alexa Fluor 488) antibodies. The assays provided a strong biochemical tool to measure pathogen-immunoreceptor interactions, overcoming the difficulties associated with transgenic cell lines and protein gene expression modulation experiments.

This study provides a protocol for evaluating the interaction of Mycobacterium tuberculosis with the SLAMF1 microbial sensor. The assays were conducted on human monocyte-derived macrophages using flow cytometry and fluorescence microscopy. The described tools are relevant for studying interactions between pathogens and immunoreceptors.

結核菌と免疫受容体SLAMF1との相互作用の研究のための蛍光アッセイ

The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic ...

Micro-CFU法による結核判定の迅速化

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.

著者スポットライト:結核ワクチンの有効性と抗原提示分析の効率的な評価のためのCFU決定の最適化 

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.

結核ワクチンの有効性評価のためのマイクロコロニー形成ユニットアッセイ

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 ...

マクロファージ継代に対する薬効を評価するために、ハイスループットアッセイの互換性

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の成長結核バイオフィルム

高スループット/高含有スクリーニングに適応した細胞内マイコバクテリアの定量化のための顕微鏡表現型アッセイ

マイコバクテリアの半定量的、ミディアムスループット列挙のためのチャコール寒天レサズリンアッセイの可視化

細胞内の標的阻害剤の有効性及び細胞毒性スクリーニングのためのシステム

結核の宿主向け治療の前臨床試験に使用する自動培養システム

マイコバクテリアに対するハイスループット薬物スクリーニングのためのアッセイ開発

マイコバクテリアに対するハイスループット薬物スクリーニングのためのアッセイ開発

マイコバクテリアに対するハイスループット薬物スクリーニングのためのアッセイ開発

結核菌と免疫受容体SLAMF1との相互作用の研究のための蛍光アッセイ

結核ワクチンの有効性評価のためのマイクロコロニー形成ユニットアッセイ