Name: 'bioluminescent' reporter phage for the detection of category a bacterial pathogens
Uploaded: 2011-07-08T12:00:00.000+00:00
Duration: 11 min 31 s
Description: Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

この研究は国立衛生研究所（NIH）の中小企業イノベーション研究プログラム（NIAID、1R43AI082698 - 01）と食糧農業のUSDA国立研究所（NIFA、2009-33610-20028）によってサポートされていました。

（1）Y.ペストのプレートの接種<ol><li>ストリークY.ペスト A1122（BeiResources＃NR15）ストック培養ルリア-ベルターニ（LB）寒天（ミラー）上に。無菌操作を使用し、すべてのYを行うクラスII型バイオセーフティーキャビネット。Y.におけるペストの操作ペスト A1122はバイオセーフティレベル（BSL）2除外選択エージェントの株である。それは、プラスミド75キロバイトのペア低カルシウム応答（LCR）病原性の両方を欠いている、と病原性に必要なPGMの軌跡。 Yを拡大28℃ ペスト ℃の空気インキュベーターを制御する静的な温度で48時間。 Yの成長率ペスト菌は 1.25 H 9の世代時間と遅いです。 </li></ol> （2）Y.ペスト菌の液体培地接種<ol><li>単一のYを転送する滅菌金属接種ループを使用してLBブロス2 mLを含有する滅菌17 × 100mmの培養チューブにペスト菌のコロニー。サイズの均一なコロニーを選択し、他のコロニーを示している、と明らかにプレート上のコロニーの残りの部分から分離されている。軽くボルテックスチューブと28で文化を育てる° C振盪インキュベーター（225 rpm）で約20時間。 </li></ol> （3）Y.ペスト菌の伸長、レポーターファージ加え、そして生物発光検出<ol><li>一晩Yを薄めるペスト菌の培養50mlファルコンチューブに新鮮なLBブロス（例えば、培地の9.5 mLに細胞を500μL）に1:20 ° C（225 rpm）で振とうしながら28で成長する。 0.2の600 nm（A 600）での光学密度（約5時間）に達するまで成長する。生物発光反応を形質導入するファージの能力は、宿主細菌の生存と適応度に相関しているので、活発に増殖する細胞は、検出のために好ましい。それにもかかわらず、検出システムは、成長のサイクル7のすべての段階で採取された細胞との互換性であることが示されている。 </li><li> 2パーセントのn -デカナール溶液を調製することにより生物発光反応に必要な基質を生成する。 LBブロスの9.8 mLでのn -デカナール200μLを混合。 5秒間激しくボルテックスプライム2％のn -デカナール溶液2mLを持つマイクロプレートルミノメーターインジェクタ。ルミノメーターはよくして、各プレートにデカナールソリューションの67μLをautoinject直ちに10秒間のサンプルを読み込むようにあらかじめ設定さ</li><li> 3文化のチューブにLBブロス1 mLのアリコートを分注する。各チューブにレポーターファージストック溶液（5 × 10 9プラーク形成単位の株式[PFU] / mL）を20μLを追加し、ファージとメディアだけではサンプルは、ネガティブコントロールとして機能します。 Yの不在でペスト菌の細胞は、レポーターファージの追加により、生物発光反応を引き出すしないでください。 </li><li> Y. 1mLのアリコートを分注する6培養管にペスト菌の培養（ステップ3.1から）。文化の3（試験培養）にレポーターファージストックの20μLを追加。残りの3の文化は&quot;単独で細胞のネガティブコントロール（バックグラウンドのautobioluminescence）として機能する。手短にボルテックスで文化をミックスし、各サンプルから収穫200μL（時間0読み取り用）と白色96ウェルマイクロタイタープレートに分注する。 28で、残りの培養液を、Cを（225 rpm）で振盪しながら。 </li><li>マイクロプレートルミノメーターを用いて生物発光の時間0サンプルを（相対発光ユニット、RLU）を測定します。 </li><li> 10以降の各サンプルの収穫200μL、20、30、40、50、および60分後のレポーターファージのほか、生物発光用の試料を測定する。 Y.場合ペスト菌の細胞が存在し、レポーターファージは、特に、細胞に結合し、そのDNAを注入し、luxABレポーター遺伝子（図1）転写と翻訳するの転写と翻訳機構をホストが使用されます。 </li><li>信号の強さと信号の応答時間が存在する細胞の数に比例します。細胞（10 5〜10 8 CFU / mL）を、対照と比較したRLUの大幅な増加が高濃度で20分以内に明らかにする必要があります。低細胞濃度では、（10 2 -10 4 CFU / mL）を、RLUの著しい増加は40〜60分以内に明らかにする必要があります。 </li><li>また、Y.を必要とせずに検出プロセスを迅速化するペスト菌の伸長は、新鮮な成長プレートからコロニーを、レポーターファージを保有するLBブロス200μLのを1.5mlのエッペンドルフチューブに直接（ボルテックス）混合することができる。マイクロタイタープレートのウェルに細胞/ファージ混合液を分注する。 28℃でマイクロタイタープレートをインキュベートし、60分（シングル時のポイントのみ）後の生物発光のためにサンプルをお読みください。 </li></ol> 4。代表的な結果： Yのレポーターファージを介した検出の代表的なタイムコース実験ペスト菌を図2に描かれている。 1の陰性コントロール）ファージ単独で（無細胞）、または2のみ）セル（無ファージ）60分株式会社を通じて約20 RLUの生物発光のベースラインレベルを提供ubation（ベースラインのレベルはルミノメーター固有のもの）。対照的に、テストサンプル（レポーターファージや細胞）のための生物発光の増加は、ファージ添加後15分で明らかである。信号強度は、60分かけて徐々に増やす必要があります。長期期間（80分以​​上）のためのインキュベーションは、宿主細胞の媒介溶解をファージに起因するピーク信号から減少する信号となります。同様の結果が37のインキュベーションの温度で得られる° Cにもかかわらず、Yの最適温度ペスト菌の成長は28℃で9です。 <img src="/files/ftp_upload/2740/2740fig2.jpg" alt="図2"/> 図2。 Yのファージを介した生物発光検出ペスト 。0の時点で、レポーターファージ、細胞を28℃でインキュベートし、混合し、そして生物発光は（RLU）を経時的にモニターした。 RLUの大幅な増加は、（*学生t -検定、p &lt;0.05）が15分以内に明らかである。

<ol>
	<li>Darling, R. G., Catlett, C. L., Huebner, K. D., Jarrett, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threats+in+bioterrorism.+I:+CDC+category+A+agents.">Threats in bioterrorism. I: CDC category A agents.</a> Emerg Med Clin North Am. 20, 273-309 (2002).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory manual of plague diagnostic tests.. , (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (1999).</li><li>Inglesby, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthrax+as+a+biological+weapon,+2002:+updated+recommendations+for+management.">Anthrax as a biological weapon, 2002: updated recommendations for management.</a> JAMA. 287, 2236-2252 (2002).</li><li>Schuch, R., Fischetti, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+genomic+analysis+of+the+Wbeta+and+gamma+phages+infecting+Bacillus+anthracis:+implications+for+evolution+of+environmental+fitness+and+antibiotic+resistance.">Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.</a> J Bacteriol. 188, 3037-3051 (2006).</li><li>Garcia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+Yersinia+pestis+bacteriophage+phiA1122+reveals+an+intimate+history+with+the+coliphage+T3+and+T7+genomes.">The genome sequence of Yersinia pestis bacteriophage phiA1122 reveals an intimate history with the coliphage T3 and T7 genomes.</a> J Bacteriol. 185, 5248-5262 (2003).</li><li>Schofield, D. A., Molineux, I. J., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+bioluminescent+phage+for+detection+of+Yersinia+pestis.">Diagnostic bioluminescent phage for detection of Yersinia pestis.</a> Journal of Clinical Microbiology. 47, 3887-3894 (2009).</li><li>Schofield, D. A., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage-mediated+bioluminescent+detection+of+Bacillus+anthracis.">Phage-mediated bioluminescent detection of Bacillus anthracis.</a> Journal of Applied Microbiology. 107, 468-478 (2009).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CDC: Basic laboratory protocols for the presumptive identification of Yersinia pestis. , 1-19 (2001).</li><li>Gunnison, J. B., Larson, A., Lazarus, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+between+Pasteurella+pestis+and+Pasteurella+pseudotuberculosis+by+action+of+bacteriophage.">Rapid differentiation between Pasteurella pestis and Pasteurella pseudotuberculosis by action of bacteriophage.</a> J Infect Dis. 88, 254-255 (1951).</li><li>Lazarus, A. S., Gunnison, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Action+of+Pasteurella+pestis+Bacteriophage+on+Strains+of+Pasteurella,+Salmonella,+and+Shigella.">The Action of Pasteurella pestis Bacteriophage on Strains of Pasteurella, Salmonella, and Shigella.</a> J Bacteriol. 53, 705-714 (1947).</li><li>Sergueev, K. V., He, Y., Borschel, R. H., Nikolich, M. P., Filippov, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+detection+of+Yersinia+pestis+using+amplification+of+plague+diagnostic+bacteriophages+monitored+by+real-time+PCR.">Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.</a> PLoS One. 5, e11337-e11337 (2010).</li></ol>

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このメソッドは、急速にYを検出するレポーターファージの能力を示していますレポーターファージからペスト菌は、培養Yに生物発光シグナルの応答を伝達することができるファージの添加後20分以内ペスト菌の細胞。レポーターファージはまた、直接Yを検出することができる分離とその後の栽培7の前提条件なしに臨床マトリックスにおけるペスト 、。一般的に完了するための48時間を必要とする標準的なファージの溶解アッセイと比較して、これは大幅に検出する時間が短縮されます。 これまでの研究では、野生型ΦA1122のファージは、ほぼすべての自然なYを溶解できることが実証されているペスト菌の分離株、 および Yのための&quot;特定&quot;です。 ペスト 6,10,11、しかし、いくつかY.偽結核の菌株は20℃6,10,12以上の温度で増殖させた場合ΦA1122敏感であることが示されている。温度に敏感な微分磁化率の理由は不明だが、細胞表面の層/組成の温度依存性の変化におそらく起因する。したがって、レポーターファージの検出システムの潜在的な注意点は、近縁種であるY.から株による偽陽性反応の可能性です。 偽結核 。制限温度（20℃）でレポーターファージアッセイを実行すると、Yを含む可能性のある試料中の偽陽性シグナルを防ぐことができます偽結核 。特定の温度で成長させた単離した培養液を使用する場合はこのように、特異性は厳密にコントロールすることができます。 Yの要約、迅速な検出と診断にペストは 、ペスト以来、正予後、特に肺ペストのために不可欠であり、治療が発症後最初の24時間以内に投与されていない場合は、ほとんど常に致命的です。このメソッドは、培養分離株の確認同定または臨床的に関連の行列内でこれらのニーズを満たす可能性を秘めています。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td> Difco社製LB寒天、ミラー</td><td> VWR </td><td> 90003-346 </td></tr><tr><td> Difco社製LBブロス、ミラー</td><td> VWR </td><td> 90003-350 </td></tr><tr><td> 17 × 100 mmの培養管</td><td>米国サイエンティフィック</td><td> 1485-0810 </td></tr><tr><td> N -デカナール</td><td>シグマ</td><td> D7384 </td></tr><tr><td> VERITASマイクロプレートルミノメーター</td><td>ターナーバイオシステムズ</td><td> 9100-001 </td></tr><tr><td>マイクロライトマイクロタイター96ウェルプレート</td><td> VWR </td><td> 62402-984 </td></tr></tbody></table>

'bioluminescent' reporter phage for the detection of category a bacterial pathogens

ペスト菌と炭疽菌は、それぞれ1、ペストや炭疽の原因物質である細菌性病原体カテゴリです。両疾患&quot;の自然発生は、今は比較的まれですが、生物兵器としてこれらの病原体を使用して、テロリスト集団の可能性は本当のです。病気の本質的な伝達性、急速な臨床経過、及び高い死亡率のため、発生を迅速に検出することが重要です。したがって、迅速な検出と診断を提供する方法論は、公衆衛生対策と危機管理の活性化の即時実施を確保するために不可欠です。 組換えレポーターファージは、Yの検出のための迅速かつ特異的なアプローチを提供することがありますペストとB.炭疽菌。疾病管理予防センターは、現在、これらの細菌性病原体2-4の確認同定のために古典的なファージの溶解アッセイを使用してください。これらのアッセイは、自然に彼らの細菌のホストの特定および溶解性であるファージを発生し活用する。特定のファージの存在下で培養細菌の一晩増殖した後、プラーク（細菌の溶解）の形成は、細菌のターゲットの肯定的な識別を提供する。これらのアッセイは堅牢ですが、彼らは3つの欠点に苦しむ：1）彼らは、実験室ベースで、2）彼らが疑われる試料から細菌の分離培養を必要とし、3）彼らは、完了までに24〜36時間かかります。これらの問題に対処するため、組換え&quot;光-タグ&quot;レポーターファージは遺伝的にYのゲノムにビブリオharveyi luxABの遺伝子を統合して設計されたペストとB.炭疽菌の特定のファージ5-8。結果luxABレポーターファージが急速にすることで、特定のターゲットを検出することができた（分以内）と敏感に受容細胞に生物発光表現型を付与する。重要なのは、検出が培ってきたレシピエント細胞にまたはモック感染臨床検体7とどちらが得られた。 デモンストレーションの目的で、ここでは知られているYのファージを介した検出のための方法を説明します。 ペスト菌は CDCペスト診断ファージΦA11226,7（図1）から構成さluxABレポーターファージを用いて分離する。軽微な変更（成長温度やメディアの例：変更）、と同様の方法は、Bの検出に使用されることがあります炭疽菌はB.を使用して隔離炭疽菌のレポーターファージWβ：：luxAB 8。方法は、栽培Yにbiolumescent表現型のファージを介した情報伝達を説明しますその後、マイクロプレートルミノメーターを用いて測定しているペスト菌の細胞。伝統的なファージの溶解アッセイに比べてこの方法の主な利点は、使いやすさ、迅速な結果、そして96ウェルマイクロタイタープレートフォーマットで同時に複数のサンプルをテストする機能です。 <img src="/files/ftp_upload/2740/2740fig1.jpg" alt="図1"/> 図1。検出の回路図。ファージを試料と混合され、ファージが細胞に感染する、luxABが発現し、細胞のbioluminescesされています。サンプルの処理が不要です。ファージや細胞を混合し、続いて光を測定している。

Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

優先細菌性病原体の同定のための簡単​​な方法は、遺伝子組み換えレポーターファージを使用することです。その特定の宿主種に固有のこれらのレポーターファージは、、急速に宿主細胞に生物発光信号応答を伝達することが可能です。ここで、我々は、検出のためのレポーターファージの使用について説明します。<em&gt;ペスト菌</em&gt;。

カテゴリの検出細菌性病原体のための&quot;生物発光&quot;レポーターファージ

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

bioluminescent-reporter-phage-for-detection-category-bacterial

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

13.4K ビュー.  Guild Associates, Inc.. 優先細菌性病原体の同定のための簡単​​な方法は、遺伝子組み換えレポーターファージを使用することです。その特定の宿主種に固有のこれらのレポーターファージは、、急速に宿主細胞に生物発光信号応答を伝達することが可能です。ここで、我々は、検出のためのレポーターファージの使用について説明します。ペスト菌。

ビデオ: 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

マウスでイメージ細菌感染にルシフェラーゼを用いた

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

免疫・感染学

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

その場で測定と細胞密度の相関、発光細菌の発光

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

生化学

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

抗生物質耐性ブドウ球菌の細菌の検出のためのバイオセンサー

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

生物工学

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

感染した組織の細菌の遺伝子の規則を勉強する蛍光ベースのメソッド

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

遺伝学

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

蛍光性DNAzymesを使用した細菌の検出

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物発光細菌のイメージング<emインビボで&gt;</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the F&ouml;rster radius, commonly less than 14 nm, FUEL can occur at distances up to &micro;m or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.

BRET歩：バインドされていない励起による蛍光発光から（燃料）

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵襲体内生物発光システムによってマウスのホストである腸管出血性大腸菌の植民地化の検出

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

化学的に媒介種間相互作用を検出するための共培養を使用して

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

生物学

Fluorescent Reporter 定量による全臓器病原体追跡(英語)

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

著者スポットライト:組織における抗生物質と免疫系に対する細菌の応答を解明する

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

微生物感染症の高解像度三次元全臓器断層撮影法

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

カテゴリの検出細菌性病原体のための"生物発光"レポーターファージ

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