私たちは、Prを感謝します。有用な技術アドバイスやアメーバ共培養し、アメーバの濃縮についての興味深い議論についてベルナールラ·スコラ。我々はまた、我々の研究室で技術を実施する中で彼の助けのために博士ヴィンセントトーマスに感謝します。

1。アメーバの共培養 1.1サンプル調製<ol><li> 環境試料 <ol><li> 水サンプル 0.22μmの孔サイズの膜を通して水サンプル（1 L 500 ml）を絞り込む。そして、ページのアメーバ生理食塩水媒体中のPASのNaCl（120ミリグラム、 硫酸マグネシウム4mgの•7H 2 O、塩化カルシウム2•2H 2 Oの4ミリグラムのNa 2 HPO 4の142ミリグラム、およびKHの136ミリグラムで膜を振る蒸留水1 L 2 PO 4）。 </li><li> 固体サンプル 蒸留水またはPBS中の活性汚泥などの土壌や砂のサンプルおよび半固体のサンプルのような固体試料を再懸濁し、0.22μmの孔サイズの膜を介してそれらをフィルタリング。その後、PASで膜を振る。 </li><li> 高度内因性アメーバ原虫で汚染サンプル 第一の低速遠心分離（180×g）でのfoによってサンプルを沈降rは10分以上5μmの孔サイズの膜を介して濾過する。さらに1.1.1のように、上清（それぞれろ液）を処理する。 注：他の汚染除去技術は、さらに環境サンプルを除染するために使用され得る：30分間50℃で試料を加熱または酸性もしくは塩基性溶液で処理する14。 </li></ol></li><li> 臨床サンプル それらの物理化学的特性に応じて、臨床サンプルを処理する。大規模な不純物を除去するフィルタまたは遠心分離機の液体。固体試料を懸濁します。 、組織を用いるダウンスhomogeneiserまたはガラスビーズを用いて、例えば、それらを粉砕し、細胞内細菌を解放するために細胞を溶解すること。 </li></ol> 1.2アメーバ準備<ol><li> スープの準備 、プロテオースペプトン、100gの、酵母エキス10g、MgSO 4で •7H 2 4.9gのペプトン、酵母エキスおよびグルコース（PYGを含有するリッチ培地：次のメディアを調製O、クエン酸ナトリウム5gの•2H 2 O、Feの（NH 4）0.1gの2（SO 4）2•6H 2 O、KH 2 PO 4、のNa 2 HPO 4 1.97gの1.7gの•7H 2 Oグルコース45gを、蒸留水5中のLのCaCl 2 0.295 g）および、例えばアカントアメーバ種についてPAS等の非栄養培地。 </li><li> アメーバ文化 <ol><li> PYG培地30mlを含む細胞培養フラスコ中で25℃で、アメーバ（優先アメーバカステラーニ ATCC 30010またはA.多食亜目のLinc-AP1）を栽培しています。 </li><li>フラスコを激しく振盪により​​アメーバを収穫し、1,500 X gで10分間、細胞懸濁液を遠心分離。 PAS培地で2回ペレットを洗浄。 KOVAスライドに細胞を計数し、ミリリットル当たり5×10 5細胞の懸濁液を得るために音量を調整する。 </li><li>マイクロプレートにアメーバサスペンションを転送します。 12ウェル当たり1ミリリットルを使用する - と24-Wエルプレート、48ウェルプレートおよび96ウェルプレートを300μlの500μlの。 25℃で少なくとも2時間、マイクロプレートをインキュベートこれは、各ウェルの底にアメーバの沈降と添付が可能になります。 </li></ol></li></ol> 1.3共培養<ol><li> サンプル接種 <ol><li>希釈していない試料100μlを皮切りに、通常の10倍希釈系列で、1.1または1.2からのサンプルの段階希釈を2.3.3からプレートに接種。 </li><li>サンプル中の微生物が存在する可能性アメーバ芝生の上の堆積物に10分間1800×gでマイクロプレートを遠心分離する。これはアメーバによる接触や微生物の食作用を増大させる。 </li><li> 25°Cで45分間プレートをインキュベートし、新鮮なPASでメディアを置換することによりPASで三回洗浄する。または抗生物質を添加せずにPASのウェルあたり1ミリリットルを追加します（ストレプトマイシン、ペニシリン、ゲンタマイシンおよび/またはバンコマイシン）、バクテリオに応じて検索される他種。 </li><li>アメーバのシストを​​避けるために、加湿雰囲気中で32℃でマイクロプレートをインキュベートする。アメーバに侵入し、溶解する細菌の存在を検出するために、20倍の対物レンズで毎日各ウェルを観察します。 </li><li>溶解の場合は、約10 5アメーバ/ cm 2の単層に共培養液100μlを接種することによって、新鮮なアメーバにサブカルチャーを実行します。特に指定された細菌種を単離するために、これらの細菌（ レジオネラ属菌のためのすなわち BCYE寒天。）用に設計された特定の寒天培地にも接種する。 </li><li>溶解がない場合には、四から七日最初の接種後の共培養液100μlを取り、24穴プレートに900μLの新鮮アメーバ文化を接種する。アメーバの急速な溶解が細菌の増殖せずに観察された場合、ウイルスが存在する可能性があります。この場合は、0.22ミクロンで、上清をフィルター処理し、新鮮なアメーバに感染するために濾過サスペンションを使用しています。 </li></OL&gt; </li></ol> 1.4細菌の単離と特性評価<ol><li> 細菌の染色 マイクロプレート24ウェルにカバーガラス上に直接共培養を行う。培地を除去し、染色または免疫蛍光を行う。 <ol><li>修正されたロマノフスキー染色<ol><li>カバーガラスを乾燥させる。カバーガラスを固定液（2 mg / Lのファストグリーンメタノール中）で5回浸す。 </li><li>染色溶液中でカバースリップを5回浸すI（リン酸緩衝液pH 6.6で1.22グラム/ LエオシンG） </li><li>最後に、染色溶液II（リン酸緩衝液pH 6.6中1.1グラム/ Lのチアジン）でカバースリップを5回浸す。 </li><li>蒸留水でサンプルを洗浄します。乾燥させ、および顕微鏡によって観察する。 </li></ol></li><li>チールネルゼン染色<ol><li>カバーガラスを乾燥させる。チールフクシンでサンプルをカバーしています。蒸気が表示されるまで炎で色素を加熱します。 </li><li>少なくとも5分間、室温でクール蒸留水ですすいでください。 2分間のイソプロパノール中、3％の塩酸溶液でサンプルをカバーし、蒸留水ですすいでください。 </li><li>メチレンブルーで30秒間カバーし、蒸留水ですすいでください。乾燥させ、および顕微鏡22で観察します。 </li></ol></li><li>ヒメネス染色<ol><li> 10％塩基性フクシン（100ミリリットルの95％エタノール中で塩基性フクシンの10g）を100mlの4％水性フェノール及び蒸留水650mlを250mlのを混合することによって塩基性フクシンのストック溶液を調製する。使用前に48時間、37℃でインキュベートする。 </li><li>カバーガラスを乾燥させ、炎を通過させることにより固定する。新たに濾過塩基性フクシン2分間（0.1ml、10mlのMリン酸ナトリウム緩衝液、pH 7.45で塩基性フクシンストック溶液4ml）でサンプルを覆う。 </li><li>水でサンプルをすすぎ、10秒間マラカイトグリーン（蒸留水中0.8％）でインキュベートする。水で再び洗浄し、マラカイトグリーンの染色を繰り返します。再び水ですすいでください。カバーガラスを乾燥させ、Mそれをountし、顕微鏡23で観察します。 </li></ol></li><li>免疫蛍光<ol><li> 10分間、4％メタノール中で5分間インキュベートすることにより、またはパラホルムアルデヒドでカバーガラスを固定します。 </li><li> PBSで3回洗浄し、室温で溶液（5％BSA、PBS中0.1％サポニン）をブロックの中で2時間インキュベートする。 </li><li>興味のある微生物に対して産生された抗体を含むブロッキング溶液中で1時間カバースリップをインキュベートする。 </li><li> PBS中で再び3回洗浄し、一次抗体に対する蛍光団にリンクされた二次抗体で1時間インキュベート。 PBSで3回洗浄、カバースリップをマウントし、蛍光顕微鏡で観察します。 </li></ol></li></ol></li><li> PCRによるDNA検出 アメーバ共培養の100〜200μLからDNAを抽出します。例えば、マイコバクテリア24種のような目的のために、16S rRNA遺伝子または特異的なプライマーを標的ユニバーサルプライマーを用いて微生物を検出する、レジオネラ25またはクラミジア 26のメンバー。 </li></ol> 2。アメーバ濃縮 2.1。試料調製ボルテックスにより、PAS中の固体や半固体試料を懸濁します。 10分間低速（180×g）で懸濁物を遠心分離する。これは、ペレットに自由生活アメーバの濃縮を可能にする。上清をアメーバ共培養し、アメーバ濃縮21用のペレットに使用することができます。 2.2。培地調製<ol><li> PASの100ミリリットルに寒天を1.5グラムを加え、121℃で培地15分オートクレーブペトリ皿に温培地を注ぎ、室温で固化してみましょう。 </li><li> 37℃で一晩、LBまたはチオグリコール酸ブロス中の大腸菌 （ATCC 25922）を育てるPBSで2回細菌を洗浄し、PAS培地で再懸濁します。 PASで10倍に希釈し、PAS寒天プレート上でこの希釈2-3ミリリットル広げて乾燥させます。 </li></ol>板の片面に試料の滴（又はフィルタの個数）を追加し、皿の中央に線を形成するペトリ皿上を流れてみましょう。 2.4。アメーバ成長とアメーバサブカルチャー<ol><li>毎日のペトリ皿を観察します。アメーバ移行フロントが検出された場合、移行前に寒天の小片を切り出し、Eの芝生で覆われ、新鮮なNNAペトリ皿に接種大腸菌 。 </li><li>所与アメーバ株の純粋培養物を得るために、試料の純度に応じて再接種を複数回繰り返す。 </li></ol> 2.5。アメーバや細菌特性評価<ol><li>細胞をこすり落とし、PASで再懸濁します。 </li><li> （例えば、アメーバおよび配列決定のために細菌の16SrRNA増幅、RESP。18S rRNAの）DNAを抽出し、PCRおよび配列決定によりアメーバおよび/または細菌内部共生の同一性を決定する。 </li> &lt;李は&gt;新鮮アメーバに接種し、試料中に存在する可能性細菌を検出するためにアメーバの共培養を行うために、PASでこれらの掻きセルを使用。 </li></ol>

<ol>
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Microbiol. 72, 2428-2438 (2006).</li><li>Miyamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+seminested+PCR+method+for+detection+of+Legionella+species+and+its+application+to+surveillance+of+legionellae+in+hospital+cooling+tower+water.">Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of legionellae in hospital cooling tower water.</a> Appl. Environ. Microbiol. 63, 2489-2494 (1997).</li><li>Lienard, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+new+chlamydiales-specific+real-time+PCR+and+its+application+to+respiratory+clinical+samples.">Development of a new chlamydiales-specific real-time PCR and its application to respiratory clinical samples.</a> J. Clin. Microbiol. 49, 2637-2642 (2011).</li><li>Wang, Y., Ogawa, M., Fukuda, K., Miyamoto, H., Taniguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+mycobacteria+from+soils+at+an+illegal+dumping+site+and+landfills+in+Japan.">Isolation and identification of mycobacteria from soils at an illegal dumping site and landfills in Japan.</a> Microbiol. Immunol. 50, 513-524 (2006).</li><li>Corsaro, D., Pages, G. S., Catalan, V., Loret, J. F., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodiversity+of+amoebae+and+amoeba-associated+bacteria+in+water+treatment+plants.">Biodiversity of amoebae and amoeba-associated bacteria in water treatment plants.</a> Int. J. Hygiene Environ. Health. 213, 158-166 (2010).</li><li>La Scola, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Legionella+drancourtii+sp.+nov.,+a+strictly+intracellular+amoebal+pathogen.">Legionella drancourtii sp. nov., a strictly intracellular amoebal pathogen.</a> Int. J. Syst. Evol. Microbiol. 54, 699-703 (2004).</li><li>Thomas, V., Casson, N., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Afipia+and+Bosea+strains+isolated+from+various+water+sources+by+amoebal+co-culture.">New Afipia and Bosea strains isolated from various water sources by amoebal co-culture.</a> Syst. Appl. Microbiol. 30, 572-579 (2007).</li><li>La Scola, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amoeba-resisting+bacteria+and+ventilator-associated+pneumonia.">Amoeba-resisting bacteria and ventilator-associated pneumonia.</a> Emerging Infect. Dis. 9, 815-821 (2003).</li><li>Collingro, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+an+environmental+Chlamydia+strain+from+activated+sludge+by+co-cultivation+with+Acanthamoeba+sp.">Recovery of an environmental Chlamydia strain from activated sludge by co-cultivation with Acanthamoeba sp.</a> Microbiology. 151, 301-309 (2005).</li><li>Lienard, J., Croxatto, A., Prod'hom, G., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrella+lausannensis,+a+new+star+in+the+Chlamydiales+order.">Estrella lausannensis, a new star in the Chlamydiales order.</a> Microbes Infect. 13, 1232-1241 (2011).</li><li>La Scola, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+giant+virus+in+amoebae.">A giant virus in amoebae.</a> Science. 299, 2033 (2003).</li><li>Boyer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+Marseillevirus+highlights+the+role+of+amoebae+as+a+melting+pot+in+emergence+of+chimeric+microorganisms.">Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 21848-21853 (2009).</li><li>Thomas, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lausannevirus,+a+giant+amoebal+virus+encoding+histone+doublets.">Lausannevirus, a giant amoebal virus encoding histone doublets.</a> Environ. Microbiol. 13, 1454-1466 (2011).</li><li>Raoult, D., Renesto, P., Brouqui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+infection+of+a+technician+by+mimivirus.">Laboratory infection of a technician by mimivirus.</a> Ann. Internal Med. 144, 702-703 (2006).</li><li>Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parachlamydia+acanthamoebae,+an+emerging+agent+of+pneumonia.">Parachlamydia acanthamoebae, an emerging agent of pneumonia.</a> Clin. Microbiol. Infect. 15, 18-28 (2009).</li><li>Lamoth, F., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amoebal+pathogens+as+emerging+causal+agents+of+pneumonia.">Amoebal pathogens as emerging causal agents of pneumonia.</a> FEMS Microbiol. Rev. 34, 260-280 (2010).</li><li>Lienard, J. G., Ashbolt, K., Sen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+6.">Ch. 6.</a> Environmental microbiology, current technology and water applications. , 143-162 (2011).</li><li>Boughalmi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+isolation+of+giant+viruses+of+the+Mimiviridae+and+Marseilleviridae+families+in+the+Tunisian+environment.">High-throughput isolation of giant viruses of the Mimiviridae and Marseilleviridae families in the Tunisian environment.</a> Environ. Microbiol. , (2012).</li><li>Ovrutsky, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooccurrence+of+Free-Living+Amoebae+and+Nontuberculous+Mycobacteria+in+Hospital+Water+Networks,+and+Preferential+Growth+of+Mycobacterium+avium+in+Acanthamoeba+lenticulata.">Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks, and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata.</a> Appl. Environ. Microbiol. 79, 3185-3192 (2013).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

アメーバ共培養及びアメーバ濃縮は、多くの新しい細菌やアメーバ種の単離を可能に効率的な方法である。これらの方法で得られた結果は、環境中で、最も興味深いことに、このような塩素化及びオゾン処理等の化学処理によって制御されると考えられる人工水中ネットワークにおけるアメーバとアメーバ耐バクテリアの両方のユビキタス存在を確認する。アメーバ共培養及びアメーバ濃縮?...

分子診断の出現する前に、環境ニッチや臨床サンプル中に存在する微生物は、多くの場合、主にペトリ皿に寒天上に、別の選択培地上で培養することによって検出された。細菌コロニーとその代謝活性の表現型は、その後、種レベルで細菌の分類を可能にした。ブロスはまた、検出の感度を増加させるために使用されてもよい。しかし、両方の技術は、これらすべてのメディアにゆっくりか成長した細菌の回復を認めていない。これは、分子のアプローチは非常に広く、最近使用されている理由です。それにもかかわらず、DNAの検出は、細菌の生存には何の手掛かりを提供しません。また、逆に文化に、分子のアプローチをさらに特徴づけることができるひずみを生じない。 成長する固体培地またはその必要性細胞にあまり増殖する病原体を研究することは複雑である。これらの「成長させることは困難」細菌のほとんどは気難しいINTRです無細胞、細菌、頻繁に発見され、それがレジオネラ·ニューモフィラの場合と同様に大規模な流行、以下を特徴とする。この細菌は米国在郷軍人会の大会中に発生した発生後特徴付けられた。できるだけ多く182のような人が感染していたと29は、重症肺炎の1,2に死亡した。それは後にアメーバがこの細菌の自然宿主であることを示したし、そのホテルの空調システムにおけるプレゼンスと水ネットワークは、いわゆるレジオネラ病3の発生の原点にあったことだった。 アメーバは、世界中に存在し、土壌、大気、水、（4に概説されている）ヒトのボランティアの鼻粘膜から単離した。これらの「自由生活」アメーバは一般的な環境で自律的に分裂しているが、時折許容宿主5に侵入することがあります。 HYによる食作用およびその後のリソソーム消化を通して様々な微生物にアメーバフィードdrolases 6。多くの通性または偏性細胞内細菌は、消化に抵抗し、このように感染し、例レジオネラ 、 クラミジアに関連した細菌やマイコバクテリアに関してはアメーバに分ける（7に見直され、8）することができます。自由生活アメーバは、おそらくまだ発見されていない細胞内細菌のための重要な潜在的な貯水池を表しています。これはローザンヌに異なるグループは、様々な環境試料9月15日から、いくつかの新しい偏性細胞内微生物を分離するために許可されたアメーバの共培養及びアメーバ濃縮と呼ばれる二つの主要な技術を実装するために私たちのグループを主導した。 アメーバは細菌に放牧専門の食細胞であることから、食作用に抵抗し、これらの原生生物の内部で増殖することができる細菌は、人間の食細胞にコロニーを形成し、人間に向けた病原性かもしれません。これは、部分的なWaddlia町などの一部クラミジア関連細菌のために証明されたndrophila。 W·chondrophilaはアメーバではなく、哺乳動物の上皮細胞、マクロファージ、および魚類細胞株16〜18のようないくつかの細胞型だけでなく成長することができます。アメーバ共培養も大きく異なる細菌種21で汚染されているスツールを含む臨床サンプル19,20、細胞内細菌を検出するための関連が表示されます。 無菌メディアにアメーバの（b）の成長と大腸菌の細菌ローン上および（C）の選択および特性、ここで我々は、環境や臨床サンプルの（a）の治療を含む、アメーバの共培養及びアメーバ濃縮の主な手順を説明します細胞内細菌の。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Glucose monohydrate</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>108342</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m pore size membrane</td>
			<td>Merck Millipore, Darmstadt, Germany</td>
			<td>SCVPU11RE</td>
			<td></td>
		</tr>
		<tr>
			<td>proteose peptone</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>211693</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>353135</td>
			<td></td>
		</tr>
		<tr>
			<td>Kova slide</td>
			<td>Hycor, Indianapolis, IN</td>
			<td>87144</td>
			<td></td>
		</tr>
		<tr>
			<td>cell culture microplates</td>
			<td>Corning Inc, Corning, NY</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>Diff-Quik staining kit</td>
			<td>Siemens Healthcare diagn., Munich, Germany</td>
			<td>130832</td>
			<td></td>
		</tr>
		<tr>
			<td>Ziehl fuchsin</td>
			<td>Fluka, St-Louis, MI</td>
			<td>21820</td>
			<td></td>
		</tr>
		<tr>
			<td>basic fuchsin</td>
			<td>Sigma, St-Louis, MI</td>
			<td>857843</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma, St-Louis, MI</td>
			<td>P1037</td>
			<td>Corrosive and mutagenic</td>
		</tr>
		<tr>
			<td>malachite green oxalate</td>
			<td>Fluka, St-Louis, MI</td>
			<td>63160</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution</td>
			<td>Electron Microscopy Sciences, Hatfield, PA</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma, St-Louis, MI</td>
			<td>84510</td>
			<td></td>
		</tr>
	</tbody>
</table>

discovery of new intracellular pathogens by amoebal coculture and amoebal enrichment approaches

このようなレジオネラ、マイコバクテリアおよびクラミジア様生物などの細胞内病原体は、彼らは多くの場合、全く通常の細菌を培養するために使用されている選択培地上で悪いか成長しないために分離することは困難である。このため、これらの病原体の多くは、ごく最近になって発見されたり、重要な発生を、以下の通りであった。これらの病原体は、しばしば、宿主細胞として機能し、細​​菌の生存および増殖を可能アメーバ、関連付けられている。アメーバ共培養及びアメーバ濃縮：私たちは、臨床的または環境試料中の細胞内病原体の単離および特徴が存在できるように2つの技術のデモを提供するために、ここにいきたいと考えています。アメーバ共培養は、試料中に存在する細胞内細菌に感染して溶解することができるアメーバの芝生の上に調査したサンプルを接種することにより、細胞内細菌の回収を可能にする。アメーバ濃縮は、臨床的または環境試料中のアメーバの回復が存在できます。目だけでなく、これらのアメーバで特異的に成長している新たな細胞内細菌の新たなアメーバ種の発見につながることができている。一緒に、これら2つの技術がアメーバに増殖することができる新たな細胞内細菌を発見するのに役立ちます。なぜならアメーバ感染し、食作用に抵抗するそれらの能力のために、これらの細胞内細菌はまた、マクロファージによる貪食を逃れる可能性があり、従って、高等真核生物に対して病原性である。

アメーバ共培養及びアメーバ濃縮を使用して、環境、および/ ​​または病原性細菌の全範囲（ 表1）に発見された。 アメーバ共培養は、環境試料、水処理プラント、水分配システムを分析するために我々のグループ等で使用されていた。広範囲の微生物は、この技術を用いて単離することができた。アメーバ共培養により単離し、最も一般的な細菌は、水?...

Watch this Scientific Journal Video about Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches at JoVE.com

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

アメーバ共培養は、選択的アメーバおよびマクロファージなどの食細胞に耐え細胞内病原体を成長させるために接着性アメーバを用いた細胞培養系である。したがって、新たな感染性病原体を発見するための重要なツールを表しています。アメーバ濃縮は、新たアメーバ種とそれらの特異的な細胞内細菌の発見を可能にする。

アメーバ共培養し、アメーバ濃縮アプローチによって新しい細胞内病原体の発見

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

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JoVE Journal

Immunology and Infection

14.4K ビュー.  University Hospital Center and University of Lausanne. アメーバ共培養は、選択的アメーバおよびマクロファージなどの食細胞に耐え細胞内病原体を成長させるために接着性アメーバを用いた細胞培養系である。したがって、新たな感染性病原体を発見するための重要なツールを表しています。アメーバ濃縮は、新たアメーバ種とそれらの特異的な細胞内細菌の発見を可能にする。 

ビデオ: Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

細胞内細菌性病原体を持つゼブラフィッシュ胚の感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

免疫・感染学

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

細胞内病原体によって引き起こされる液胞破裂のシングルセル測定

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

メンブレン極性膜完全性、細胞内イオン濃度の監視の変更<em&gt;肺炎球菌</em&gt;蛍光染料を使用

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

細胞内細菌によって遠藤-リソソームシステムの再構築を検討するために、蛍光ナノ粒子の応用

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

日和見菌病原体への蛍光タグ付き遺伝子のバイオリスティック変容<em&gt;クリプトコックス·ネオフォルマンス</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

16S rRNAの遺伝子配列決定およびMALDI-TOF MSによるレア細菌性病原体の同定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

ナノスケールPCRによる動物標本における呼吸器病原体のハイスループット検出

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

蛍光寿命イメージングによる細胞内のスネアを可視化

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

ジカ特定診断エピトープの探索

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

病原体、共生生物、およびホストの腸上皮間の相互作用を研究する人間 Colonoid 単分子膜

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...