作品は、国立衛生研究所と米国肺協会からの財政支援を行った。

1。 Mの成長バイオフィルム250mlのスクリューキャップボトルの結核 <ol><li>メディアの準備：水900mlの中のグリセロールの60mlを、クエン酸第二鉄アンモニウムの0.05グラムのMgSO 4 KH 2 PO 4、0.5グラム、L-アスパラギン4グラム、クエン酸2gの、の0.5グラムに溶解する。 NaOHでpHを7.0に調整します。実験を始める直前に涼しいし、オートクレーブでは、最終濃度0.1％w / vのに滅菌硫酸亜鉛4を追加MC 2 7000パントテン酸要求株であるため、この株は、最終濃度10μg/mlのでパントテン酸を必要とします。 </li></ol> 注：これは、結核のために使用Sautonのメディアの標準的な組成物である。ただし、必要に応じてその他の特殊なメディアは、他のマイコバクテリア種に使用することができます。 <ol start="2"><li>接種の準備：M.グロウ 0.7の一週間で0.05％のTween-80、またはOD 600 1.0〜7H9OADCで結核 。 cuはltureは直接1:100希釈で接種物として使用することができます。 </li><li> 250mlのスクリューキャップ付きポリスチレン瓶（コーニング）にSautonのメディアの25MLを分注する。非常に緊密に培地、キャップボトルに接種の250μlのを追加し、3週間加湿37℃インキュベーターに邪魔されずにそれを置く。全く汚染がないことを確認するために毎日1回瓶を観察します。 </li><li>第三週の終わりに、Mの更なる成長を可能にするためにボトルのキャップを緩め界面での結核 。キャップがこの段階で緩んでいないされている場合、コンテナ内の不十分な酸素濃度は、細菌の更なる成長を遅らせることになります。 </li></ol> 2。 M.の成長12ウェルプレート中の結核バイオフィルム<ol><li>セクションA1とA2で説明したようにMC 2 7000のメディアと接種を準備します。 </li><li>接種の600μlでメディアの60mlを混合します。 4.5メートルディスペンスプレートの各ウェルに混合物のL。蓋付きプレートをカバーしています。パラフィルムでプレートを数回巻き付けます。 5週間37℃で加湿インキュベーター内で邪魔されずに、プレートをインキュベートします。 </li></ol> 3。 M.における薬剤耐性の永続化の頻度を決定する結核のバイオフィルム<ol><li> M.を育てるセクションBで説明したように、12ウェルフォーマットでの結核のバイオフィルムはこれが約5週間の合計になります。 </li><li>バイオフィルムが成熟された後（インキュベーションの5週間後）にピペットでマイクロチップを用いたバイオフィルムの下にメディアに所望の濃度で抗生物質の選択を注入します。 </li></ol>注：ペリクルの下にメディアのボリュームは約3.0mLまで減少します。したがって、研究者はそれに応じて薬の量を計算する必要があります。 <ol start="3"><li>スワール抗生物質を徹底的に培地中に拡散して静かになるようにプレート。統計的に有意な結果を得るためには、抗生物質を注入する4ウェルの耳。並行して、抗生物質が他の4つのウェルに溶解した溶媒と同じボリュームを注入し、手付かずのプレートの最後の4つの井戸を残す。蓋とカバープレートとプレートの周りにパラフィルムの新鮮な層を配置します。希望の期間のインキュベーターに戻します。 </li><li>インキュベーションの終わりに、プレートを開き、各ウェルに0.1％（体積/体積）の最終濃度になるようにトゥイーン-80を追加します。渦巻洗剤の均一分散のために静かにプレート全体。 15分間室温で静置します。その内容全体が均一に15mlのコニカルチューブに移すことができるので、ピペットで数回、各ウェルの内容を混ぜ合わせます。 </li><li>室温で10分間4000RPMでチューブの内容をスピンダウンする。新鮮な洗浄緩衝液（10％グリセロール、0.05％のTween-80を含むPBS）5mLのでペレットを再懸濁します。洗濯を3回繰り返します。洗浄緩衝液の5mLのでペレットを再懸濁します。 Oのためにロッカーに保管してください4でvernight℃、 </li></ol> 注意：低温は、もともとM.スメグマチスのために開発（分散時に、その成長を最小限にするため）と同様に結核のために使用されていますが、成長の遅い種が最も可能性の高い結果に影響を与えることなく、室温で揺れることができます。 BSL-3施設で作業している場合、室温でロッキングする必要がある可能性があります。 <ol start="6"><li> 、適切なサイズに、その幅広いエンドをカットするシリンジにフィットし、パラフィルムでそれをラップすることによって、マイクロチップ（2-200μlの）を装備し、滅菌注射器を準備します。先端フィット注射器からチューブの内容全体を渡して、新鮮な15mlのチューブに集める。あなたはかなり均一な懸濁液を観察するまで、このステップを5から6回繰り返します。 </li><li>各ウェル中の生存コロニーの数を決定するために7H11OADCプレート上に懸濁液およびプレート希釈液の希釈系列を準備します。 37℃インキュベーターで3週間培養する。周波数を決定する溶剤処理されたプレート上で得られたものと扱われ、抗生物質で得られたコロニー数の比を計算することにより、バイオフィルムの人口の永続化機構ち消されます。 </li></ol> 4。代表的な結果 M.の瓶で培養し、成長結核は、最初の週の終わりまでにボトルの底で見ることができます。エア·メディア·インターフェイスでの成長は第三週（図1A）の末尾に常に表示されているものの、2週目の終わりまでに、エア·メディア·インターフェイス上の細菌の斑状の成長は、見ることができます。この時点では容器の壁への細菌の付着も観察されています。この時点以降の文化の成長は主にエア·メディア·インターフェイス上で発生します。表面成長の下に液体が明確である。通常、構造体は、5週目（図1B）の終わりまでに成熟する。インキュベーションが長引く場合には、構造体は、容器の底に沈むが開始されます。興味深いことに、第三週の終わりまでキャップの締め付けは、プロセスの重要なステップであり、未知の理由のためにルーズに覆われた瓶は大幅にインターフェイス9上での成長の開始を遅らせる。 12ウェルフォーマットでは、空気媒体の界面で強固なバイオフィルムは、5週間（図2A）の最後にウェルのそれぞれで見られます。プレートは完全にラップされていない場合は、差動、バイオフィルムの成長が観察される。最悪の場合には、重要なメディアの蒸発は、細菌の増殖（図2B）を停止させることができます。したがって、プレートのラッピングは、両方の蒸発を防ぐためだけでなく、バ​​イオフィルム形成（前の段落を参照）環境を提供する必要があります。 この手法で決定バイオフィルム中の生菌数は非常に再現性がある。 M.の応答結核バイオフィルムは抗生物質の性質によって異なります。そのようなリファンピシンとして殺菌性抗生物質については、生存率の低下は、バイファに従う<html> SICの傾向9。人口の割合が小さいに関係なく、抗生物質や曝露時間の濃度の抗生物質に完全に反抗的なままにする永続的な段階に続いた最初の3〜4日の間に生存率の急激な減少。図3は、リファンピシンの50μg/mlの7日間暴露（MICの50倍）の後に成熟したバイオフィルム中の生菌数を示します。 <img alt="図1A。" src="/files/ftp_upload/3820/3820fig1A.jpg" /> M.の図1A。早期出現インキュベーションの3週間後の細菌のエア·メディア·インターフェイス上で結核菌 。 <img alt="1Bを示しています。" src="/files/ftp_upload/3820/3820fig1B.jpg" /> 図1Bは、Mのバイオフィルムを成熟インキュベーションの5週間後の空気·メディア·インターフェイス上で結核 。 820/3820fig2A.jpg &quot;/&gt; M.の図2A 5週齢のバイオフィルム結核は、12ウェルフォーマットで増殖させた。 <img alt="図2B。" src="/files/ftp_upload/3820/3820fig2B.jpg" /> 図2B。パラフィルムなしで12ウェルプレートに結核菌バイオフィルムの成長に失敗した。 <img alt="図3。" src="/files/ftp_upload/3820/3820fig3.jpg" /> M.における薬剤耐性の永続化の頻度を示す図3代表的なプロット結核は、12ウェルフォーマットで成長し、7日間のリファンピシン50μg/mlのにさらされたバイオフィルム。

<ol>
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我々は、開示することは何もありません。

結核菌の感染によって引き起こされる結核（TB）は、世界の公衆衛生にとって大きな脅威となっている。世界の人口の3分の1近くが無症候病原体に感染すると推定され、約900万の新しいケースは、結核や感染症の約170万、ダイの症状は毎年11で毎年診療所に表示されます。病気の大きな負担には、主にワクチンと六から九ヶ月にわたって投与多剤併用療法を含む非常に複雑な化?...

<table border="1">
	<tbody>
		<tr>
			<td>Equipment and supplies</td>
			<td>SUPPLIER</td>
			<td>CATALOG NUMBER</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>Model # 1923/25</td>
		</tr>
		<tr>
			<td>Polystyrene culture bottles</td>
			<td>Fisher Scientific</td>
			<td>03-374-300</td>
		</tr>
		<tr>
			<td>12-well tissue culture plate</td>
			<td>VWR</td>
			<td>62406-165</td>
		</tr>
		<tr>
			<td>50-mL conical tubes</td>
			<td>VWR</td>
			<td>89039-660</td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>Thermo Scientific</td>
			<td>57019-662</td>
		</tr>
		<tr>
			<td>Chromatographic refrigerator</td>
			<td>VWR</td>
			<td>55702-520</td>
		</tr>
		<tr>
			<td>petri dish</td>
			<td>VWR</td>
			<td>25384-342</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>REAGENT</td>
			<td>SUPPLIER</td>
			<td>CATALOG NUMBER</td>
		</tr>
		<tr>
			<td>KH2PO4 (monobasic)</td>
			<td>EMD</td>
			<td>PX1565-1</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher</td>
			<td>M65-500</td>
		</tr>
		<tr>
			<td>L-asparagine</td>
			<td>Sigma</td>
			<td>A4284-100G</td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sigma</td>
			<td>C1857-100G</td>
		</tr>
		<tr>
			<td>ferric ammonium citrate</td>
			<td>Sigma</td>
			<td>F5879-100G</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>EMD</td>
			<td>GX0185-5</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045-500G</td>
		</tr>
		<tr>
			<td>ZnSO4</td>
			<td>Sigma</td>
			<td>Z4750-500G</td>
		</tr>
		<tr>
			<td>D-pantothenic acid</td>
			<td>Sigma</td>
			<td>P2250-25G</td>
		</tr>
		<tr>
			<td>Difco Middlebrook 7H9 Broth</td>
			<td>Becton Dickinson</td>
			<td>271310</td>
		</tr>
		<tr>
			<td>Middlebrook OADC Enrichment</td>
			<td>BBL</td>
			<td>212351</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher</td>
			<td>T164-500</td>
		</tr>
		<tr>
			<td>250mL storage bottle</td>
			<td>Corning</td>
			<td>430281</td>
		</tr>
		<tr>
			<td>12 well plates</td>
			<td>Falcon (BD)</td>
			<td>353043</td>
		</tr>
		<tr>
			<td>rifampicin</td>
			<td>Sigma</td>
			<td>R3501-1G</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>J.T. Baker</td>
			<td>9070-05</td>
		</tr>
		<tr>
			<td>10mlLsyringe</td>
			<td>Becton Dickinson</td>
			<td>301604</td>
		</tr>
		<tr>
			<td>1-200&mu;L pipet tips</td>
			<td>VWR</td>
			<td>89079-458</td>
		</tr>
		<tr>
			<td>parafilm M</td>
			<td>VWR</td>
			<td>PM-996</td>
		</tr>
		<tr>
			<td>15mL centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>188-285</td>
		</tr>
		<tr>
			<td>Difco Mycobacteria 7H11 Agar</td>
			<td>Becton Dickinson</td>
			<td>283810</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-1</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333-500G</td>
		</tr>
		<tr>
			<td>Na2HPO4 (dibasic)</td>
			<td>Sigma</td>
			<td>S0876-500G</td>
		</tr>
	</tbody>
</table>

growth of mycobacterium tuberculosis biofilms

結核菌は、ヒト結核の病原体は、抗生物質を含む環境ストレスに対して生き残るためには並外れた能力を持っています。ただしMのストレス耐性結核は、結核の1 6ヶ月の長期化学療法への可能性が高い貢献の一つであり、病原体のこの特徴的な表現型の根底にある分子メカニズムは不明である。多くの微生物種がバイオフィルム2から4と ​​呼ばれる高度に組織化され、接続された表面、およびマトリックスカプセル化された構造に自己組織化によるストレスの多い環境で生き残るために進化してきました。コミュニティの成長は、微生物の好適な生存戦略であることが表示され、表面の付着、細胞間コミュニケーション、細胞外高分子物質の合成（EPS）5,6を制御する遺伝的要素によって実現されます。環境ストレスに対する耐性はおそらくEPSによって、おそらくphysioloによって促進されるバイオフィルム7の複雑なアーキテクチャ内で異種の微小環境に個々の菌のgical適応。 最近の一連の論文では、確立されているM.結核と結核スメグマチスは、抗結核薬イソニアジドとリファンピシン8-10 50回以上最小発育阻止濃度を許容できるバイオフィルムと呼ばれる組織的な多構造で成長する強い傾向を持っています。M.結核は、しかし、興味深いことにヘッドスペースの特定の午前9時01分比で、成熟したバイオフィルムを形成するために特定の条件を満たす必要があります。メディアのほか、大気9空気の限られた交換。特殊な環境条件の要件は、おそらくそのM.実際にリンクすることができます結核は絶対ヒト病原体であり、したがって、組織の環境に適応している。この文書では、培養M.ための方法を実証する結核ボトルや細菌などの遺伝子研究のための便利な12ウェルプレートフォーマットでのバイオフィルム。我々は、M.の弱毒株のためのプロトコルを説明した病原体9のin vivo増殖に重要である2つの遺伝子座、panCDとRD1、欠失を持つ結核、MC 2 7000、。この株は、安全に、高価なBSL-3施設の要件を回避し、結核の病原体の基礎生物学を理解するためのBSL-2の封じ込めに使用することができます。メソッドは、他の培養可能なマイコバクテリア種のバイオフィルムを成長させるために、メディアの適切な変更で、拡張することができます。 全体的に、培養抗酸菌バイオフィルムの均一なプロトコルは、マイコバクテリアの基本的な弾力性のある特性の研究に興味を持って捜査に役立ちます。さらに、成長マイコバクテリアバイオフィルムの明確かつ簡潔な方法は、臨床や製薬INVを助けるestigators潜在的な薬剤の有効性をテストします。

Watch this Scientific Journal Video about Growth of Mycobacterium tuberculosis Biofilms at JoVE.com

Growth of Mycobacterium tuberculosis Biofilms

特定の条件下で培養すると結核菌は、薬剤耐性バイオフィルムを形成します。ここでは、培養M.ための方法を説明結核のバイオフィルムと薬剤耐性の永続化の頻度を決定する。これらのプロトコルは、M.の薬剤耐性のメカニズムにさらなる研究のために有用であろう結核 。

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 ...

growth-of-mycobacterium-tuberculosis-biofilms

Research

JoVE Journal

Immunology and Infection

Growth of Mycobacterium tuberculosis Biofilms

23.6K ビュー.  University of Pittsburgh. 特定の条件下で培養すると結核菌は、薬剤耐性バイオフィルムを形成します。ここでは、培養M.ための方法を説明結核のバイオフィルムと薬剤耐性の永続化の頻度を決定する。これらのプロトコルは、M.の薬剤耐性のメカニズムにさらなる研究のために有用であろう結核 。

ビデオ: Growth of Mycobacterium tuberculosis Biofilms

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

マイクロタイタープレート形式で液体希釈法により、第一、第二ラインの薬のための結核菌の複合体の抗菌薬感受性試験

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

免疫・感染学

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.

のサンプル調製<em&gt;結核菌</em核磁気共鳴代謝研究のための&gt;抽出

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

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.

自然送信時に結核マラリア重複感染を研究する実験モデル結核菌とマラリアベルゲイ</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.

多剤耐性を実証<em&gt;結核菌</em&gt;増幅マイクロアレイ

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

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

回復への新規マイクロダイセクションアプローチ<em&gt;結核菌</em肺肉芽腫、ホルマリン固定パラフィン包埋から&gt;特異的転写

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

上の機能的研究のための3Dヒト肺組織モデル<em&gt;結核菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

歯科バイオフィルムにおける細胞外のpHのレシオメトリックイメージング

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

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 ...

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.

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 ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

18FDG PET/CT イメージング勉強結核感染と非ひと霊長類における治療のためのツールとしての解析

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...