<html> 「陽性」として1。血液培養ブロス旗<ol><li>継続的な監視のインキュベーションキャビネットから合図血液培養ボトルを取り外し、生物学的安全キャビネットに入れてください。注：ボトルは、危険な微生物を含むことができ、ユニバーサル予防措置に従わなければする必要があります。サンプリングによるエアロゾル中の感染リスクに、すべてのサンプリング手順は、バイオセーフティクラスII層流キャビネット内で実行する必要があります。 </li></ol> 2。グラム染色を用意する<ol><li>地元の制度的なプロトコルに従って合図血液培養液からグラム染色を準備します。注：グラム陰性生物は、顕微鏡で同定されている場合、血液培養液を以下の方法に従って処理される。グラム陽性菌が特定される場合には、遺伝的同定と耐性マーカーを対象とした代替分子法は（この記事で扱われていない）培養液13に適用されます。 </li></ol>血清分離管にフラグ付きの血液培養ブロスのE &quot;&gt; 3。転送<ol><li>ゆっくりと2〜3倍を反転させることにより血液培養ボトルを混ぜる。 </li><li>バイオセーフティキャビネット内での安全性の血液搬送装置に10ミリリットルの注射器を取り付けます。 </li><li>血液培養ボトルに血液転送装置を取り付け、注射器にスープの5ミリリットルを撤回。血清分離チューブに吸引BCブロスを転送します。 </li></ol>血液培養ブロスの4。濃度<ol><li>赤血球の大容積を除去する15分間1,250×gで血清分離管を遠心分離する。 </li><li>吸引し、直ちにゲル/流体界面の上に軟膜の約1ミリリットルを残すように注意して、無菌トランスファーピペットを用いて上清を捨てる。注：好気性ボトルはゲル/流体界面が、不透明な白色に現れると、チューブの底部に赤血球の明確な分離を示す。対照的に、時ゲル/流体界面に起因上清中に懸濁したままで溶解した赤血球成分に深赤色で表示され、溶解性嫌気性血液培養ブロスに適用した。 </li></ol> 5。遠心洗い手順を繰り返します<ol><li>優しく滅菌ピペットでゲル界面上軟膜流体の最後の1ミリリットルを混合し、次いで1.5mlのマイクロチューブにボリューム全体を転送します。 30秒288×gで新しいチューブを遠心分離する。 </li><li>新しい1.5 mlマイクロチューブにプラスチックの使い捨て1ミリリットルのホールピペットを用いて上清を移し、ペレットとチューブは捨てる。注意：これは注意がペレットの転送を避けるために注意され、この段階で重要である。これは上清色素沈着残り、ペレットを常に明確に示されていないように嫌気紀元前ボトルから処理されている標本のために特に重要です。 </li></ol> 6。残留細胞の溶解を</p&gt; <ol><li> 1分間遠心した後、吸引（および廃棄）のための18407×gで試料ペレットを中断することなく、可能な限り少ない残液を残して細かいな先端のホールピペットを用いて上清。 </li><li>上下にピペッティングにより無菌DNaseおよびRNA分解酵素を含まない水1ミリリットル中の残留ペレットを再懸濁。注：一部の著者は、非生存細菌をレンダリングペレットを再懸濁するために滅菌水の代わりにアルコール溶液の使用が記載されている。 </li><li> 1分間18407×gで再懸濁ソリューションを遠心分離する。 </li></ol>細菌タンパク質の7。抽出<ol><li>吸引し、再び、できるだけ多くの液体が除去されることを確実に先の細いピペットで吸引を使用して、上清を捨てる。 </li><li> 10μlのギ酸（70％v / v）の中にペレットを再懸濁。注：それが吸入される場合は、それが皮膚と接触するとギ酸が身体に影響を与えることができる。溶液を調製するか、操作するとき、それはrとスタッフは個人用保護具の他に、ヒュームのキャビネットを使用することをecommended。 </li><li>均質な再懸濁ソリューションを確実にするためにピペットを用いてよく混ぜる。物理的にペレットを破壊するためにピペットチップを使用すると、より均質な溶液を確保するために必要であり得る。 </li></ol> MALDI-TOFターゲットプレート8の調製<ol><li>ターゲットスポットあたり調製した懸濁液の2μlでクリーンなMALDI-TOFターゲットプレートに接種。読み込み失敗の時々の問題を克服するために、各サンプルの3標的部位を準備します。 </li><li> MALDI-TOFターゲットプレートを乾燥させる。乾燥速度は、プレートを横切る空気の流れを最大にするために、ヒュームフードの端に板を配置することによって増加させることができる。 </li><li>マトリックス溶液（10 mg / mlのα-シアノ-4 - ヒドロキシ桂皮酸（HCCA）、50％アセトニトリル、2.5％トリフルオロ酢酸）1μlを各々乾燥させたスポットを重ねる。注：それが吸入されている場合、またはそれがコンタに入ってくる場合は、マトリックス溶液が体に影響を与えることができる皮膚とCT。溶液を調製するか、操作するときには、スタッフが個人用保護具の他に、ヒュームのキャビネットを使用することをお勧めします。 </li><li>前述したように、MALDI-TOFターゲットプレートはヒュームフードの端に乾燥させます。均質な製剤は、プレート上の標的スポットのそれぞれを満たすことを確認してください。 </li></ol>質量分析計に9か所、MALDI-TOFターゲットプレート<ol><li> MALDI-TOF質量分析計へのMALDI-TOFターゲットプレートを挿入する。プレートはスプリング式のプレートと同一面に座っていることを確認します。注意：ターゲットのすべてのスポットが乾燥するまで、ターゲットプレートを挿入しないでください。 </li><li>ゴム製のシールが必要な真空状態を作成することができますができるように糸くず、ほこりや髪の毛の汚れていないことを確認してください。 </li><li> MALDI-TOFステージふたを閉めてから、機器の前面にある「IN / OUT」ボタンを押してください。蓋は現在ロックして、必要な真空が作成できるようになります。 </li></ol> 10。MALDI-TOF MS Spectra買収<ol><li> MALDI BiotypingとFlexControlソフトウェアを起動します。 </li><li>タイピングソフトウェア内の「ファイル」というタイトルのドロップダウンメニューから「新しい分類」を選択します。プロジェクトに名前を付けてから、「新規作成」を選択します。タイピングソフトウェア内のセットアップを完了するために、「次へ」を選択します。 </li><li>制御ソフトウェアのウィンドウ内でターゲットプレートの画面上のグラフィックを識別します。マウスの左ボタンを押しながら接種したスポットの上にマウスを移動して使用されているMALDI-TOFターゲットスポットを強調表示します。 </li><li>選択された目標位置の任意の場所をクリックし、ドロップダウンメニューから「検体を追加」を選択し、マウスの右ボタンを使用してください。 </li><li>対象スポットのそれぞれについて、「ID」欄に血液培養識別の詳細を追加、完了したら「次へ」をクリックします。 </li><li>商業「分類」スペクトルのデータベースを選択し、「次へ」を選択します。注意：ラボratoriesは、基準スペクトルの数を増加させるため、社内の追加または商用データベースを利用することができる。 </li><li> 「完了」をクリックし、MALDI-TOF MSは、スペクトルを生成開始します。注：MALDI-TOFの設定は、製造者の設定（リニアポジティブモード、60Hzのレーザー周波数、20kVの加速電圧、16.7 kVのをIS2電圧は、170ナノ秒の抽出遅延、および2,000-20,137 m / z範囲）あたりの通りであった。 </li><li> MALDI制御ソフトウェアはピークを生成するために失敗した場合は、スペクトルの手動取得する（メソッドが記述されていない）を行ってもよい。 </li></ol> MALDI-TOF MSスコアの11。ポスト解析の解釈<ol><li>スペクトルを取得し、分析が完了したら、各ターゲットの識別リストを展開しタイピングソフトウェア、種の識別の横の「+」ボタンを選択します。 </li><li>トップ5が類似している場合には注意して、トップ5の同定を研究。スコア同じターゲットの≥1.7と不調和トップ5属スポットは、混合ブロスの可能性を提起した。注：MALDI-TOF技術は、混合ブロスの検出のための貧弱な感度を有する。たとえば、培養液が混合種を含む場合に、高い信頼度スコアは、混合種のほんの一例について同定され得る。 </li><li>スコア≥2.0がターゲット場で最高の試合のために注意されると、種を報告している。 </li><li>ときにターゲットスポットが≥1.7での最高スコアが&lt;2.0のみ属を報告します。トップ5識別の追加基準が一致している場合には、種レベルに報告する。 </li></ol> 12。MALDI-TOFターゲットプレートの削除を<ol><li>すべてのスペクトルは、MALDI-TOFマシンの「IN / OUT」ボタンを押して完了している場合。 </li><li> MALDI-TOFターゲットプレートレセプタクルを開いて、MALDI-TOFターゲットプレートを取り外します。 </li><li>アルコールを使用して、クリーンなすべてのターゲットスポットを再利用可能な、MALDI-TOFターゲットプレートを使用している場合、それを清潔で乾いたら、RTで、MALDI-TOFプレートを格納します。 </li></ol>

<ol>
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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。

ポストの遠心操作は分離された成分をリミックスしない十分な注意を払って行われた血液培養液にMALDI-TOF MSを適用するときには重要です。これは、MALDI-TOFスペクトルに干渉スパイクを生成することができる、ヘモグロビンを含む血液培養成分およびヒト細胞タンパク質を除去することが特に重要である。 MSは種のための≥2.0のスコアのカットや属同定のための≥1.7を推?...

原因細菌に血流感染（BSI）の患者は、6〜48％1に至るまで、高い院内死亡率を持ち続ける。適切な経験的抗生物質の送達は、生存を促進し、重症敗血症の患者のサブセットでは、適切な治療への各時間の遅延が生存2,3の低下と相関する。従って、臨床検査室の主な目的は、迅速に検出、同定および臨床決定を通知するために、血液培養物中の細菌の存在を伝えることである。これは、微生物学研究室は、グラム染色4を報告し、最近時の抗菌療法に最も大きな影響を与える、観察研究は、飛行時間型質量分析法（MALDI-TOF MS）のマトリックス支援レーザー脱離/イオン化時間を実証したことが実証されている血液培養ブロス上で直接実行グラム陰性菌5に起因するBSIの三分の一以上で処方に影響する。 6,7の同定のための有効な実験室ツールにつながっている。技術は、現在十分に確立されており、固体培地の6,8で単離した微生物の迅速かつ正確な同定のための多くの研究所に統合されました。血液培養のための低コストでの微生物の初期の識別を取得する可能性の両方の臨床医や研究室の管理者への微生物のアピールのために「陽性」合図している（BC）ブロスにMALDI-TOF MSの直接適用。 血液培養ブロスへのMALDI-TOF MSの直接適用の臨床的有用​​性は、47から98.9の範囲のグラム陰性細菌の同定の成功の報告と、識別の標準的な表現型の培養ベースの方法と比較した場合に観察される感度の広い範囲によって制限されてきた％9月11日 。感度の変動性が高いBCブロス組成物、初期細菌濃度、サンプル調製法の変動ならびに試験集団9において遭遇するグラム陰性生物体のアレイに関する。これらの他の公開されたプロトコルと比較して、ここで提示した方法は、エタノール、塩化アンモニウムまたは追加の（非マトリックス）アセトニトリルの使用を回避する。その結果、細菌ペレットは、潜在的な表現型感受性試験法は培養液中で、これらの生物に直接適用することを可能にする（タンパク質抽出の時点まで）の実行可能なままになります。また、提示された方法は、時間12最小限の&#39;実践&#39;で、血液培養グラム染色結果の25分以内に利用可能な細菌同定と、安価で信頼性が高く、迅速であることが示されている。 この方法では、ギ酸抽出を利用した単純な社内スピン溶解プロトコルは、グラム負のbを同定するために陽性の血液培養液に直接適用さMALDI-TOF MS技術とacteria。

<table border="0" cellpadding="0" cellspacing="0" width="1319">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">BACTEC Plus Aerobic/F Medium</td>
			<td style="width:307px;">Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right" style="width:188px;">442192</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Lytic/10 Anaerobic /F Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442265</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Peds Plus Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442194</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer - Blood transfer device</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">364880</td>
			<td>Single use sampling device reducing the risk of needlestick injury</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer SST 5.0mL, Advance plus</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">367954</td>
			<td>Serum separating tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe 10 mL</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">302143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Samco, USA</td>
			<td>222-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette (fine tipped)</td>
			<td>Samco, USA</td>
			<td>232-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge tube (2.0 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030.120.094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile water (DNAse and RNAse free)</td>
			<td>Life Technologies, Carlsbad, California, USA</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix solution&#160;</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td align="right">285074</td>
			<td>10 mg/ml &#945;-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Benchtop microflex LT MALDI-TOF MS</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td></td>
			<td>Utilizing BioTyper 3.1 (Build 65) and FlexControl 3.3 (Build 99) software</td>
		</tr>
	</tbody>
</table>

rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry

臨床微生物学研究室の重要な役割は、血流感染を引き起こす細菌の迅速な同定を提供することです。伝統的な識別は、固体寒天上のコロニーが成熟した後でのみ利用可能な識別を合図血液培養液の継代培養を必要とします。 MALDI-TOF MSは、固体培地上のコロニーに適用される臨床的に関連する大部分の細菌の同定のための信頼性のある、迅速な方法である。それは、細菌の種の同定を促進し、臨床管理を改善する可能性を有するとして直接血液培養ブロスへのMALDI-TOF MSの適用は魅力的なアプローチである。しかし、克服すべき重要な問題は、除去されない場合、MSスペクトルに干渉し、不十分なまたは低判別識別スコアをもたらすことができる血液培養検体中に含まれる樹脂の妨害、タンパク質、およびヘモグロビンの分析前の除去である。それに加えてバクターを濃縮する必要があるIA十分な品質のスペクトルを開発する。提示された方法は、濃縮、精製、及び合図血液培養液からの細菌の早期発見を可能にし、グラム陰性菌の抽出について説明します。

生成されたMALDI-TOF MSスペクトルは、スペクトルの集積参照データベースと比較される。対数スコアは、試験分離株の間の一致の信頼のために割り当てられ、参照データベースは分離し、種への可能性を識別するための属レベル（ 図1）と≥2.0に足りる相当の識別（ 図に必要なスコア≥1.7の勧告にされている2）。スコア≥1.7と入力ソフトウェアで一致した...

Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

直接血培養液への航空券のマトリックス支援レーザー脱離/イオン化時間（MALDI-TOF）質量分析（MS）の適用は、細菌の同定を促進。提示した方法は、血液培養ブロスから直接、グラム陰性細菌の同定のための迅速かつ信頼できる方法である。

MALDI-TOF質量分析法を使用した血液培養ブロスからのグラム陰性菌の迅速な同定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

rapid-identification-gram-negative-bacteria-from-blood-culture-broth

Research

JoVE Journal

Immunology and Infection

24.1K ビュー.  Westmead Hospital. 直接血培養液への航空券のマトリックス支援レーザー脱離/イオン化時間（MALDI-TOF）質量分析（MS）の適用は、細菌の同定を促進。提示した方法は、血液培養ブロスから直接、グラム陰性細菌の同定のための迅速かつ信頼できる方法である。 

ビデオ: Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

の識別のための1.5時間の手順腸球菌</em直接血液培養から>種

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

免疫・感染学

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

ホット水 - フェノール抽出によるグラム陰性菌からの精製とリポ多糖の可視化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

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 ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

全細胞MALDI-TOF質量分析は、マクロファージ活性化の異なるモードを分析するための正確かつ迅速な方法である

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

腎臓からの二重否定αβT細胞の分離

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

迅速な細菌同定および抗生物質感受性試験のための血液培養ペレットの調製

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

ダルババンシンの参照ブロス微量希釈法<em&gt;インビトロ</em好気的に成長する細菌の&gt;感受性試験

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

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 ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

マクロファージコロニー刺激因子分化したヒト単球由来マクロファージの文化

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

デキストランは、マウスとヒト NK 細胞のレンチ ウイルスの伝達効率を向上します。

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...