These experiments have been supported by research grants from Merck and Co. and Summit, PLC.

異なる環境または臨床源からC.ディフィシルを単離する<ol><li>環境分離物：0.85％NaClで軽く濡らした滅菌済み綿ゲージを使用して、関心領域（床、ドア、ハンドル、棚など ）の表面を拭きます。 8滅菌手袋を着用し、綿棒を滅菌済みのチューブに入れてください。 </li><li>臨床分離株（便）：接種ループを使用してセフォキシチン - サイクロセリン - フルクトース寒天（CCFA）上に10〜100mgの臨床便検体をプレートし、厳重な嫌気条件下で48〜72時間インキュベートする。さらなる分析のために、 CryovialsにC.difficileストックの分離したコロニーを-80℃で保存する。 7,9,10 </li><li> 0.05％タウロコール酸ナトリウムを含むブレインハートインフュージョン（BHI）ブロスの環境スワブサンプルを濃縮し、37℃の嫌気性チャンバーに入れます5日間。 10,000×gで1mLの培養液を遠心分離し、100μLのエタノールにペレットを再懸濁する。 </li><li>再懸濁した細胞（50μL）をシクロセリン・エキシチン・フルクトース寒天（CCFA）プレート上にプレートし、37℃で40〜48時間嫌気性チャンバーでインキュベートする。さらなる分析のために、 CryovialsにC.difficileストックの分離したコロニーを-80℃で保存する。 </li><li>ラテックス凝集試薬またはPCRを用いて疑わしいクロストリジウム・ディフィシル（C.difficile）コロニーを試験する。 </li></ol> C.ディフィシルの培養およびキネティクス手順の殺傷<ol><li> 37℃で48時間、嫌気性チャンバー内の血液寒天プレート上の精製された環境的または臨床的C.ディフィシレ株を増殖させる。 </li><li>接種ループで1つの単離コロニーをとり、15 mLチューブで5 mL BHI培地に移し、37℃の嫌気性チャンバーで24時間増殖させます。 </li><li>前培養1：100を約10 6コロニー形成単位0.1％タウロコール酸ナトリウムおよび適切な濃度の抗生物質（T0）を補充した新鮮な予備還元BHIS（BHI + 5g / L酵母抽出物および1％L-システイン）中でのs（CFU）/ mL。 </li><li>各時点（T0、T6、T24、T48）でピペットを用いて1mLのサンプルを採取し、少量（100μLを連続希釈したもの）を血液寒天プレート上に撒き散布する。細胞を37℃の嫌気性チャンバー内で48時間血液寒天プレート上で増殖させ、得られたコロニー数をカウントしてCFUを決定する。 </li></ol>走査型電子顕微鏡のための試料の準備<ol><li>マイクロ遠心チューブの各時点から1 mLの細胞を集め、10,000 xgで10分間遠心分離します。上清を捨て、PBSで細胞を洗浄する。 </li><li> 10,000xgで10分間再度サンプルを遠心分離し、上清を捨てる。 1mLの4％パラホルムアルデヒドで細胞を再希釈し、室温で1時間インキュベートする。 </li><li>サンプルを再び1分間10分間遠心分離する。0,000xgで上清を捨てる。細胞を蒸留水で2回洗浄し、100μLの蒸留水で再希釈する。溶液の濁度に応じて容量を調整する。 注：複数の連続希釈を行うことは良い考えです。 </li><li>カバースリップにラベルを貼り、40μLのサンプルを加えます。フローフード内で15分間インキュベートして液体を蒸発させ、細胞をカバーグラスに接着させる。液体がまだ残っている場合は、ブロワーを使用して液体を取り除いてください。 </li><li>机上のスパッタリング装置に細胞をラベルしたカバースリップを置き、テープを剥がします。スパッタリング装置に純金を固定します。機械をオンにし、低圧（50mTorr）でスパッタリングを開始する。 80nmで30秒間被覆し、20nmの金コーティングとなる。 </li><li>コーティングされた細胞を走査電子顕微鏡に移す。 </li></ol> 4.走査電子顕微鏡上のC.ディフィシレ細胞の画像化<ol><li>走査電子顕微鏡（SEM）を適切に通気するコンピュータソフトウェアのベントボタンを押す。 </li><li>カーボンテープを使用して、コーティングされたカバーガラスを金属ステージに固定します。 SEMが通気されると、ドアは容易に開くはずです。金属ステージをねじ込むことでSEMチャンバーに固定します。 </li><li>コンピュータソフトウェアのPUMPボタンをクリックします。 SEMが &quot;Vac OK&quot;と表示されているときにSEMを使用できます。 </li><li> DetectorタブのSE検出器をクリックします。電圧を表示するボタンをクリックしてビームをオンにします。電圧を上げる前に、より低い電圧（5 kV）でイメージングを開始します（最大15 kV）。ビームがオンになった後に画像が表示されます。 </li><li>トラッキング機能を使用して、コーティングされたカバースリップの画像を描く領域を見つけます。領域を拡大し、棒状の構造を見つける。これらはクロストリジウム・ディフィシルである。 </li><li>システムを較正するために、画像を拡大して焦点を合わせる。これは、複数の作業距離（15,9および5 mm）で行う必要があります。 5mmの作動距離で画像。 </li><li>あなたのスイッチを入れて高解像度のイメージングモードを開始し、非点収差の焦点合わせと最適化を開始します。 </li><li>高倍率でピントを合わせ始める。これを行うには、粗くて細かいフォーカストグルを使用します。非点収差も同様に調整して、より鮮明な画像を得る。 <ol><li>高倍率で非点収差を調整する。これを行うには、非点収差トグル（微妙な/粗いフォーカストグルに似ています）を調整し、コンピュータソフトウェアをデジタルズームして画像を明瞭に確認します。 </li></ol></li><li>スロースキャン機能を使用して高品質の画像を収集します。収集した画像を解析に使用する.TIFFファイルとして保存します。分析中に測定が行われる場合は、データバーが選択されていることを確認してください。 </li><li>角度を変えて画像を収集する（手動で角度をSEMで直接回転させることで最大52°まで）角度のついた画像はより深い情報を示す傾向があります。 </li><li>撮影されている細胞に応じてビーム電圧を変更します。これは、ほとんどの場合、5〜15 kVの間で行います。 </li&gt; <li>イメージングが完了したら、ビームをオフにし、作動距離を20 mmに上げます。その後、チャンバを通気することができ、ステージを取り外すことができる。さらに分析するために、すべての画像をドライブにコピーします。 </li></ol> 5.画像処理と解析<ol><li>自由に利用できるソフトウェアFIJI（http://fiji.sc）をコンピュータにダウンロードしてインストールします。 </li><li> FIJIで画像ファイルを開きます。 </li><li>ライン機能を使用して、スケールバーを正確にトレースします。 </li><li> FIJIプログラムのAnalyzeタブをクリックし、最後にSelect Scaleを選択します。スケールバーに基づいて既知の距離の設定が必要なウィンドウが表示されます。長さの単位も変更し、[OK]をクリックします。 </li><li>プログラムが距離に対して校正されたので、ライン関数を使用してセルの長さを測定します。長さを取得するには、line関数を使用して、セル全体をトレースします。 [分析]タブを再度選択し、[測定]をクリックします。長さはappでなければならない以前に示されたユニットの耳。 </li></ol>

<ol>
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KWG has received past and current research support from Merck &amp; Co. and Summit, PLC.

現在の研究の目的は、C.difficileを単離し、抗生物質の薬理学的作用のより完全な特徴付けのための手段として走査型電子顕微鏡（SEM）を用いて抗生物質感受性を試験するためのハイスループット法を作製することであった。本明細書で概説したプロトコールを用いて、抗生物質治療に対する細胞の表現型応答を画像化することにより、薬物の薬理学的作用についての洞察を明らかにすること?...

Clostridium difficileはグラム陽性の胞子形成細菌であり、米国では毎年約50万の感染を引き起こし、最高レベルのリスクである疾病防除センター（CDC）による脅威レベルの緊急の病原体と考えられています。 1過去10年間に、クロストリジウム・ディフィシレ（C.difficile）に対する抗菌剤の開発が著しかった。 インビトロ研究は、薬物開発プロセスの必要な要素です。伝統的に、 インビトロ感受性および時間殺滅試験は、将来の動物および他のインビボ研究を検証するために使用される。 これらの方法は殺傷作用を評価するために重要な役割を果たすが、薬理学的治療に対する細胞の表現型応答を捕捉しない。スタンドで走査型電子顕微鏡（SEM）を組み込むことにより動力学研究を殺すと、抗生物質の直接的効果のより完全な特徴付けが可能である。ここでは、SEMを抗生物質治療の有効性をプロファイルする手段として使用する方法を提示する。

<table><tbody><tr><td>cotton gauze&amp;nbsp;</td><td>Caring</td><td>PRM21408C</td><td></td></tr><tr><td>NaCl</td><td>Macron</td><td>7532</td><td></td></tr><tr><td>50 mL tubes</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>Brain Heart Infusion (BHI)&amp;nbsp;</td><td>Criterion</td><td>C5141</td><td></td></tr><tr><td>L-cysteine</td><td>Alfa Aesar</td><td>A10389</td><td></td></tr><tr><td>yeast extract</td><td>Criterion</td><td>C741</td><td></td></tr><tr><td>sodium taurocholate</td><td>Alfa Aesar</td><td>A18346</td><td></td></tr><tr><td>anaerobic chamber</td><td>Coy</td><td></td><td>vinyl anaerobic chamber</td></tr><tr><td>cycloserinecefoxitin fructose agar (CCFA) plates</td><td>Anaerobe systems</td><td>AS-213</td><td></td></tr><tr><td>blood agar plates</td><td>Hardy diagnostics</td><td>A-10</td><td></td></tr><tr><td>latex agglutination reagent</td><td>Oxoid</td><td>DR1107A</td><td>C. diff test kit</td></tr><tr><td>microcentrifuge tubes</td><td>Eppendorf</td><td>222363204</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010-031</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>Fisher Scientific</td><td>50-259-98</td><td></td></tr><tr><td>microscope slides</td><td>J. Melvin freed brand</td><td>7525M</td><td>75 x 25mm</td></tr><tr><td>flow hood</td><td>Labconco</td><td>Class II type A2&amp;nbsp;</td><td>biosafety cabinet</td></tr><tr><td>desk sputtering machine</td><td>Denton Vacuum</td><td>Desk II</td><td></td></tr><tr><td>tape</td><td>Plastic Core</td><td>05072-AB</td><td>SPI Double Sided Adhesive Carbon Tape</td></tr><tr><td>gold</td><td>Denton Vacuum</td><td>TAR001-0158</td><td>2.375&amp;rdquo; Diameter x .002&amp;rdquo; Thick Gold foil</td></tr><tr><td>scanning electron microscope</td><td>FEI</td><td>XL-30</td><td></td></tr></tbody></table>

a protocol to characterize the morphological changes of clostridium difficile in response to antibiotic treatment

嫌気性細菌に向けられた新薬開発による抗生物質作用の評価は困難で技術的に要求される。可能なMOAについての洞察を得るために、抗生物質曝露に伴う形態学的変化を走査型電子顕微鏡（SEM）を用いて視覚化することができる。 SEMイメージングを伝統的なキル曲線と統合することで、薬物行動への洞察が向上し、医薬品開発プロセスが進歩する可能性があります。この前提を試験するために、既知の異なるMOA（バンコマイシンおよびメトロニダゾール）を有する薬物を用いて、曲線を殺し、SEM研究を行った。 C. difficile細胞（R20291）を抗生物質の存在下または非存在下で48時間まで増殖させた。 48時間間隔で、複数の時点で細胞を収集して、抗生物質の有効性を判定し、SEMで画像化した。以前の報告と一致して、バンコマイシンおよびメトロニダゾールは、コロニー形成単位（CFU）カウンターによって測定して、24時間の処理後に有意な殺菌活性を有したティン。 SEMイメージングを用いて、メトロニダゾールは、対照およびバンコマイシンと比較して、細胞長（各抗生物質について細胞長の&gt; 50％の減少; P &lt;0.05）に有意な効果を有することが判明した。薬物治療に対する表現型応答はこれまでにこのように報告されていないが、イメージングおよび測定の汎用性および信頼性ならびに他の実験化合物に対するこの技術の適用を実証する薬物のMOAと一致する。

クロストリジウム・ディフィシル（Clostridium difficile）は胞子形成細菌であるため、機能的分析の前に栄養細胞と胞子細胞との形態学的相違を決定することが不可欠である。 図1は、増殖曲線および胞子細胞の指数期の間に捕捉された栄養細胞の代表的な画像を示す。図示のように、栄養細胞は長くて滑らかな棒状の構造であるのに対し、胞子は粗?...

Watch this Scientific Journal Video about A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment at JoVE.com

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

形態変化を特徴付けるプロトコル<em&gt;クロストリジウム・ディフィシル</em&gt;抗生物質治療に対する反応

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

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Immunology and Infection

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

8.9K ビュー.  University of Houston College of Pharmacy. Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

ビデオ: A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

設計と低コストの使用、自動Morbidostat細菌の適応進化のための抗生物質選択下

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

遺伝学

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

培養およびメンテナンス<em&gt;クロストリジウム·ディフィシル</em&gt;嫌気性環境での

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

免疫・感染学

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

水系抗生物質耐性菌の単離・同定と抗生物質耐性遺伝子の分子特性評価

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

環境学

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

イメージング質量分析 による 寒天上および感染組織内で増殖した細菌コロニーの微生物連携の検討

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...

生化学

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

臨床的に関連のセフォペラゾン処理されたマウスモデル<em&gt;クロストリジウム・ディフィシル</em&gt;ひずみR20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

バイオタイプの維持と差別化に使用される実験技術<em&gt;コレラ（Vibrio cholerae）</em&gt;臨床分離株

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

蛋白質のマイクロ アレイの試金抗原特異的抗体の応答次のクロストリジウム ・ ディフィシル感染症の血清学的定量

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

経口抗生物質による治療後マウス腸内細菌叢の量的なポリメラーゼの連鎖反応に基づく分析

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

蛍光系抗生物質プローブを用いた細菌耐性の可視化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon bactericidal antibiotic treatment, the bulk of the bacterial population is rapidly killed. This first rapid phase of killing is followed by a substantial decrease in the rate of killing as the persister cells remain viable. Classically, persistence is determined at the population level by time/kill assays performed with high doses of antibiotics and for defined exposure times. While this method provides information about the level of persister cells and the killing kinetics, it fails to reflect the intrinsic cell-to-cell heterogeneity underlying the persistence phenomenon. The protocol described here combines classical time/kill assays with single-cell analysis using real-time fluorescence microscopy. By using appropriate fluorescent reporters, the microscopy imaging of live cells can provide information regarding the effects of the antibiotic on cellular processes, such as chromosome replication and segregation, cell elongation, and cell division. Combining population and single-cell analysis allows for the molecular and cellular characterization of the persistence phenotype.

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence describes the ability of small subpopulations within a sensitive isogenic population to transiently tolerate high doses of bactericidal antibiotics. The present protocol combines approaches to characterize the antibiotic persistence phenotype at the molecular and cellular levels after exposing Escherichia coli to lethal doses of ofloxacin.

大腸菌における抗生物質持続性の集団および単一細胞解析

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon ...

形態変化を特徴付けるプロトコル

要約

さらに動画を探す

この動画の章

設計と低コストの使用、自動Morbidostat細菌の適応進化のための抗生物質選択下

培養およびメンテナンス

水系抗生物質耐性菌の単離・同定と抗生物質耐性遺伝子の分子特性評価

イメージング質量分析による寒天上および感染組織内で増殖した細菌コロニーの微生物連携の検討

臨床的に関連のセフォペラゾン処理されたマウスモデル

バイオタイプの維持と差別化に使用される実験技術

蛋白質のマイクロアレイの試金抗原特異的抗体の応答次のクロストリジウム・ディフィシル感染症の血清学的定量

経口抗生物質による治療後マウス腸内細菌叢の量的なポリメラーゼの連鎖反応に基づく分析

蛍光系抗生物質プローブを用いた細菌耐性の可視化

大腸菌における抗生物質持続性の集団および単一細胞解析

形態変化を特徴付けるプロトコル

要約

さらに動画を探す

この動画の章

設計と低コストの使用、自動Morbidostat細菌の適応進化のための抗生物質選択下

培養およびメンテナンス

水系抗生物質耐性菌の単離・同定と抗生物質耐性遺伝子の分子特性評価

イメージング質量分析 による 寒天上および感染組織内で増殖した細菌コロニーの微生物連携の検討

臨床的に関連のセフォペラゾン処理されたマウスモデル

バイオタイプの維持と差別化に使用される実験技術

蛋白質のマイクロ アレイの試金抗原特異的抗体の応答次のクロストリジウム ・ ディフィシル感染症の血清学的定量

経口抗生物質による治療後マウス腸内細菌叢の量的なポリメラーゼの連鎖反応に基づく分析

蛍光系抗生物質プローブを用いた細菌耐性の可視化

大腸菌における抗生物質持続性の集団および単一細胞解析

イメージング質量分析による寒天上および感染組織内で増殖した細菌コロニーの微生物連携の検討

蛋白質のマイクロアレイの試金抗原特異的抗体の応答次のクロストリジウム・ディフィシル感染症の血清学的定量