Name: cefoperazone-treated mouse model of clinically-relevant clostridium difficile strain r20291
Uploaded: 2016-12-10T13:00:00
Description: Watch this Scientific Journal Video about Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291 at JoVE.com

The authors would like to thank Trevor Lawley at the Wellcome Trust Sanger Institute for C. difficile R20291 spores and James S. Guy at the North Carolina State University College of Veterinary Medicine for Vero cells, both utilized in this manuscript. Animal histopathology was performed in the LCCC Animal Histopathology Core Facility at the University of North Carolina at Chapel Hill, with special assistance from Traci Raley and Amanda Brown. The LCCC Animal Histopathology Core is supported in part by an NCI Center Core Support Grant (2P30CA016086-40) to the UNC Lineberger Comprehensive Cancer Center. We would also like to thank Vincent Young, Anna Seekatz, Jhansi Leslie, and Cassie Schumacher for helpful discussions on the Vero cell cytotoxicity assay protocol. JAW is funded by the Ruth L. Kirschstein National Research Service Award Research Training grant T32OD011130 by NIH. CMT is funded by the career development award in metabolomics grant K01GM109236 by the NIGMS of the NIH.

倫理的な声明： 獣医のノースカロライナ州立大学カレッジの施設内動物管理使用委員会（IACUC）（NCSU）は、この研究を承認しました。 NCSU動物管理ポリシーを使用しNCSUで1985実験動物施設の動物保護法およびヘルスリサーチ延長法に定める基準とガイドラインは、 実験動物の管理と使用に関する指針に定められたガイドラインに準拠して適用されます。動物の健康状態を毎日?...

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This protocol characterizes the clinical course, including weight loss, bacterial colonization, toxin cytotoxicity, and histopathological changes in the gastrointestinal tract, of antibiotic-treated mice challenged with C. difficile R20291 spores. There are several critical steps within the protocol where attention to detail is essential. Accurate calculation of the C. difficile spore inoculum is critical. This calculation is based on the original C. difficile spore stock enumeration, which sho...

クロストリジウム・ディフィシルは、生命を脅かす下痢1の原因となる嫌気性、グラム陽性、芽胞形成桿菌です。 クロストリジウム・ディフィシル感染症（CDI）が増加し、ヒトの罹患率および死亡率と年間1-4あたりの医療費のオーバー$ 4.8十億の結果と関連しています。 2013年には、疾病管理予防センター（CDC）は、それが公衆衛生1に緊急の脅威をもたらすことを示す、緊急の抗生物質耐性のリスクとしてクロストリジウム・ディフィシルを分類しました。現在、抗生物質バンコマイシンによる治療およびメトロニダゾールはCDI 5のためのケアの標準と考えられています。患者2,5-7の30％ -残念ながら、抗生物質による治療成功後のCDIの再発は20で発生した、高いです。したがって、この腸管病原体に対する新規治療法の発見が必要です。 ディフィシル 、交流で人間の病気を近似するモデル動物に対する治療薬を評価するために、linically関連株が必要とされています。 最初は、コッホの仮説は、クリンダマイシン処理したシリアンハムスターモデル8を使用して、1977年にC.ディフィシルのために設立されました。このモデルは、まだ病因9,10上のC.ディフィシル毒素の効果を調査するために、今日利用されています。しかし、CDIは、ハムスターモデルで高い死亡率をもたらし、CDI 10,11を有するヒトで見られる慢性潜行性の疾患の症状を近似しません。研究におけるマウスのプラットフォームのアクセシビリティと試薬可用性に基づいて、CDIのマウスモデルは関連しています。 2008年には、CDIの堅牢なマウスモデルは、クリンダマイシン12の腹腔内注射した3日間飲料水中の抗生物質カクテル（カナマイシン、ゲンタマイシン、コリスチン、メトロニダゾール、及びバンコマイシン）でマウスを処理することによって設立されました。これは、CDIと重度の大腸炎の影響を受けやすいマウスをレンダリング。左右される投与接種用量にる、臨床徴候および死亡率の範囲は、このモデルを用いて観察することができます。この時以来、様々な抗生物質療法は、 クロストリジウム・ディフィシルが消化管コロニー形成することができる点に植民地抵抗を低減、マウスの腸内細菌叢を変化させることが検討されている（ベストらに検討を。そしてローリー＆ヤング）13,14。 最近になって、5または10日間、飲料水中に与えられた広域スペクトルセファロスポリン、セフォペラゾンは、再現CDI 15感受性マウスをレンダリングします。第三世代セファロスポリンの投与は、ヒトにおけるCDIのリスク増加と関連しているので、セフォペラゾンモデルの使用は、より正確に天然に存在する病気16を反映しています。 クロストリジウム・ディフィシルの影響を受けやすいセフォペラゾン処置したマウスは、 クロストリジウム・ディフィシル胞子および臨床に至るまでの株の様々な栄養細胞の両方でチャレンジされています関連性と毒性17。感染性形態としてクロストリジウム・ディフィシレ栄養細胞を利用し、元の研究のいくつかにもかかわらず、 クロストリジウム・ディフィシルの胞子は変速機18の主要なモードと考えられています。 過去十年間では、C。ディフィシル R20291、NAP1 / BI / 027株は、CDI 19,20の流行を引き起こし、浮上しています。私たちは、セフォペラゾン処置したマウスは、R20291、臨床的に関連すると遺伝的に扱いやすいC.ディフィシル株で攻撃したとき、疾患の臨床経過を決定しようとしました。このプロトコルは、体重減少、細菌コロニー形成、毒素の細胞毒性、およびC.ディフィシル R20291の胞子でチャレンジしたマウスの胃腸管における組織病理学的変化を含め、臨床経過を詳しく説明します。全体として、このマウスモデルは、CDIは、ヒトの疾患を近似するための貴重な実験プラットフォームであることが分かります。この特徴づけマウスモデルは、このような効果を評価するために利用することができます臨床疾患の改善にとC.ディフィシルに対する植民地抵抗の回復に対する新規治療薬の。

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			<td height="21" style="height:21px;width:292px;">#62 Perisept Sporidicial Disinfectant Cleaner&#160;</td>
			<td style="width:143px;">SSS Navigator</td>
			<td style="width:119px;">48027</td>
			<td style="width:483px;">This product will require dilution as recommended by the manufacturer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22 &#956;m filter</td>
			<td>Fisherbrand</td>
			<td>09-720-3</td>
			<td>Alternative to filter plate for indivdiual samples tested in the Vero Cell Assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>Needs to be heated in water bath at 37C prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.4% Trypan Blue</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">1% Peniciilin/Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
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			<td height="21" style="height:21px;">10% heat inactivated FBS</td>
			<td>Gibco</td>
			<td>16140-071</td>
			<td>Needs to be heated in water bath at 37C prior to use</td>
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		<tr height="21">
			<td height="21" style="height:21px;">1ml plastic syringe&#160;</td>
			<td>BD Medical Supplies</td>
			<td>309628</td>
			<td></td>
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			<td height="21" style="height:21px;">1X PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
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			<td height="21" style="height:21px;">2 ml Micro Centrifuge Screw Cap</td>
			<td>Corning</td>
			<td>430917</td>
			<td></td>
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			<td height="21" style="height:21px;">96 well cell culture flat bottom plate</td>
			<td>Costar Corning</td>
			<td>CL3595</td>
			<td></td>
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			<td height="21" style="height:21px;">96 well filter plate</td>
			<td>Millipore</td>
			<td>MSGVS2210</td>
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			<td height="21" style="height:21px;">Adhesive Seal</td>
			<td>ThermoScientific</td>
			<td>AB-0558</td>
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			<td height="21" style="height:21px;width:292px;">Bacto Agar</td>
			<td style="width:143px;">Becton Dickinson</td>
			<td style="width:119px;">214010</td>
			<td>Part of TCCFA plates (see below)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Bacto Proteose Peptone</td>
			<td style="width:143px;">Becton Dickinson</td>
			<td style="width:119px;">211684</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefoperazone</td>
			<td>MP Bioworks</td>
			<td>199695</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefoxitine</td>
			<td>Sigma</td>
			<td>C47856</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clostridium difficile Antitoxin Kit</td>
			<td>Tech Labs</td>
			<td>T5000</td>
			<td>Used as control for Vero Cell Assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clostridium difficile Toxin A</td>
			<td>List Biological Labs</td>
			<td>152C</td>
			<td>Positive control for Vero Cell Assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-cycloserine</td>
			<td>Sigma</td>
			<td>C6880</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Distilled Water</td>
			<td>Gibco</td>
			<td>15230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM 1X Media</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>Needs to be heated in water bath at 37C prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fructose</td>
			<td>Fisher</td>
			<td>L95500</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td>Bright-Line, Sigma</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:292px;">KH2PO4</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">P285-500</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:292px;">MgSO4 (anhydrous)</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">M2643</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Millex-GS 0.22 &#956;m filter</td>
			<td style="width:143px;">Millex-GS</td>
			<td style="width:119px;">SLGS033SS</td>
			<td style="width:483px;">Filter for TCCFA plates&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:292px;">Na2HPO4</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">S-0876</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">S640-3</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Number 10 disposable scalpel blade</td>
			<td>Miltex, Inc</td>
			<td>4-410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR Plates</td>
			<td>Fisherbrand</td>
			<td>14230244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic petri dish</td>
			<td>Kord-Valmark Brand</td>
			<td>2900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile plastic L-shaped cell spreader</td>
			<td>Fisherbrand</td>
			<td>14-665-230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe Stepper</td>
			<td>Dymax Corporation</td>
			<td>T15469</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurocholate</td>
			<td>Sigma</td>
			<td>T4009</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrapure distilled water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:292px;">C57BL/6J Mice</td>
			<td style="width:143px;">The Jackson Laboratory</td>
			<td align="right" style="width:119px;">664</td>
			<td style="width:483px;">Mice should be 5-8 weeks of age</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Olympus BX43F light microscope</td>
			<td style="width:143px;">Olympus Life Science</td>
			<td style="width:119px;"></td>
			<td style="width:483px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DP27 camera</td>
			<td style="width:143px;">Olympus Life Science</td>
			<td style="width:119px;"></td>
			<td style="width:483px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">cellSens Dimension software&#160;</td>
			<td style="width:143px;">Olympus Life Science</td>
			<td style="width:119px;"></td>
			<td style="width:483px;"></td>
		</tr>
	</tbody>
</table>

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.

代表的な研究の間、5週齢のC57BL / 6 WTマウスは、5日間、飲料水（0.5 mg / mlで）でセフォペラゾンで前処理し、2日間は、通常の飲料水で洗い流すせました。マウスを、0日目に強制経口投与を介して、 クロストリジウム・ディフィシル R20291の10 5胞子（ 図1A）で攻撃しました。マウスは、14日間のCDIの体重減少および臨床的徴候（嗜眠、食欲不?...

Watch this Scientific Journal Video about Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291 at JoVE.com

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

このプロトコルは、臨床的に関連すると遺伝的に扱いやすい歪み、R20291を使用して、 クロストリジウム・ディフィシル感染症（CDI）のセフォペラゾンマウスモデルの概要を説明します。臨床疾患のモニタリング、 ディフィシル細菌列挙、毒素の細胞毒性、およびマウスモデルにおいてCDIを通じて組織病理学的変化を重視したプロトコルで詳述されています。

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 ...

Research

JoVE Journal

Immunology and Infection

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

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

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 ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

熱の予防によりラットに悪影響をストレス<em&gt;枯草菌</em&gt;ひずみ

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

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

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

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 ...

Influenza A Virus Studies in a Mouse Model of Infection

感染症のマウスモデルでの A 型インフルエンザ ウイルス研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

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

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

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 ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

浄化、体外(p) ppGppクロストリジウム ・ ディフィシルから合成酵素の活性

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

新生児モデル敗血症マウスの制御モデル

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

腹部大動脈瘤マウスモデルにおける脈拍伝播速度、分解性およびひずみの測定

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

血清型1肺 炎球 菌519/43株における変異体の構築

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

実験的自己免疫性ブドウ膜炎:眼内炎症性マウスモデル

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...