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.

<ol>
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The authors have nothing to disclose at this time.

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 should be consistent over multiple spore enumerations prior to starting this protocol. If the inoculum calculation is erroneous, it will result in an inaccurate inoculation dose of the mice. On the same note, preparation and dilutions of C. difficile spores based on these calculations should be prepared with care. Any dilution errors will result in an inaccurate starting spore inoculum. Lastly, precautions should be taken to avoid contamination of antibiotic-treated control mice (not inoculated with C. difficile spores). Cefoperazone-treated mice are extremely susceptible to C. difficile colonization and thus should not be in direct contact with C. difficile-infected mice28-30. If the aforementioned critical steps are recognized, successful execution of the CDI mouse model is expected. 
Modification of the dose or strain of C. difficile spores administered to the mice may alter the outcomes of this model. Increasing the spore inoculum administered to mice could increase the morbidity and mortality observed, depending on the strain of C. difficile administered. A dose response experiment, evaluating a range of several inoculum doses, is recommended if using other C. difficile strains in order to characterize the clinical course of CDI. 
This protocol uses TCCFA medium for C. difficile enumeration of spores and vegetative cells collectively. However, if distinct phases (such as germination and outgrowth) of the C. difficile lifecycle are desired, differential plating is required. Non-heat-treated contents, as described in this protocol, will yield the CFU per g content of spores and vegetative cells together. If the same GI content is heat-treated (20 min at 65 &#176;C) and then plated on TCCFA media, this will yield a CFU of spores only, since C. difficile vegetative cells are destroyed by heat. Comparison of C. difficile enumeration using these two plating techniques allows for more detailed information about the C. difficile lifecycle in vivo. 
One major limitation of this mouse model is that the results may not be reproducible in mice from different strains or vendors. Recall that disruption of the gut microbiota following cefoperazone treatment renders mice susceptible to CDI28. Variations in the initial composition of the gut microbiota between different murine strains from different vendors could alter the mouse model of CDI31. Once researchers have successfully completed this model, it is recommended that the mouse strain and vendor from where the mice are acquired stay consistent for additional studies to limit variations in the gut microbiota composition, which could alter the outcomes of the model. 
Another limitation of the protocol is that the Vero cell cytotoxicity assay does not quantitatively measure C. difficile toxin levels in vivo. Instead, epithelial (Vero) cell cytotoxicity represents a qualitative approach that measures relative cytotoxicity of C. difficile toxins present in samples. Other techniques such as qRT-PCR and immunoblotting would be helpful to determine the amount of toxin present in the samples. 
Various models of CDI are currently being used, including the Syrian hamster model, ex vivo models, and human intestinal organoid models13,14,30,32. However, the mouse model of CDI provides the increased availability of commercially-available murine reagents and assays to further examine the pathogenesis of C. difficile. Other mouse models of CDI have used different strains of C. difficile, such as VPI 10463, which results in substantial mortality and thus does not recapitulate human CDI12,17,29. In this model, C. difficile R20291 was selected for its genetic tractability and clinical relevance as a recent 027 epidemic strain33,34. Compared to other murine models of CDI, R20291 was not uniformly lethal at the dose administered, allowing for observation of a prolonged clinical course of infection concordant with human disease. Overall, this mouse model proves to be a valuable experimental platform of CDI. This fully-characterized C. difficile mouse model can be utilized to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

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>
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		<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>
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			<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>
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			<td height="21" style="height:21px;">0.4% Trypan Blue</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
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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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			<td height="21" style="height:21px;">1ml plastic syringe&#160;</td>
			<td>BD Medical Supplies</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 ml Micro Centrifuge Screw Cap</td>
			<td>Corning</td>
			<td>430917</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well cell culture flat bottom plate</td>
			<td>Costar Corning</td>
			<td>CL3595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well filter plate</td>
			<td>Millipore</td>
			<td>MSGVS2210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive Seal</td>
			<td>ThermoScientific</td>
			<td>AB-0558</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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、食欲不振、下痢、および猫背の姿勢）をモニターしました。 C.ディフィシル R20291胞子とC57BL / 6 WTマウスの課題は、24時間および48時間後にチャレンジ（ 図1B）内に有意な体重減少の中に下痢になりました。この実験では観察されなかったが、C。ディフィシル R20291の高用量でチャレンジしたマウスは、重症になったり、48で20％以上の体重減少を持つことができます- 72時間チャレンジ後、このように人道的な安楽死を必要とします。したがって、マウスは1日2回の最小値を必要としますC.ディフィシレ胞子とチャレンジ後の監視。 7チャレンジ後（ 図1B） -この代表的な研究では、マウスにおいて体重減少および疾患の臨床徴候の有意な量も日2で観察されました。 7日チャレンジ後に、マウスは体重が増加し始め、疾患の臨床症状がおさまりました。実験の最後の週では、マウスは下痢などのCDIの臨床徴候の証拠がないと、臨床的に正常に見えました。 糞は前クロストリジウム・ディフィシル胞子でのチャレンジと48時間毎、攻撃後（ 図1C）に採取しました。抗生物質治療の前に、マウスを、 クロストリジウム・ディフィシルが定着していませんでした。 クロストリジウム・ディフィシル胞子と24時間後の挑戦の中で、マウスは糞便1g当たりC.ディフィシル （ 図1C &lt;の10 7 CFUのが定着しました。/強いです&gt;）。マウスは、永続的に、実験の最後の7日間の体重減少またはCDIの臨床徴候の証拠にもかかわらず、実験を通してクロストリジウム・ディフィシルが定着したままでした。 <img alt="図1" src="/files/ftp_upload/54850/54850fig1.jpg" /> 図1：C。ディフィシル R20291展示減量にチャレンジし、 クロストリジウム・ディフィシルを保菌しているセフォペラゾン処置マウス。 A）5週齢のC57BL / 6 WT JAXマウス（1群あたりN = 3M / 3F）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、通常の飲用で洗い流すせました水。マウスを日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胞子をチャレンジした0マウスは、体重減少と14日間CDIの臨床徴候（無気力、食欲不振、下痢、および猫背の姿勢）をモニターしました。糞便は直ちにAFTE、前セフォペラゾンを開始するまでに収集し、細菌計数のために使用されましたR抗生物質を終了し、0日、1、2、3、4、5、7、9、11、13に、そして感染を通して14。剖検は、マウスがC.ディフィシル R20291でのチャレンジ後にベースライン体重のかなりの量を失った）日0、2、4、7日に行われ、14 Bました。マウスの平均体重は、0日目、ベースライン量と比較した場合、有意な体重減少が7を介して日2に見られた0日目から毎日測定しました。チャレンジ後の糞便中のC）C.ディフィシル R20291細菌負荷。 クロストリジウム・ディフィシル胞子での攻撃後24時間以内に、すべてのマウスは、糞便1g当たり10 7 CFUを（コロニー形成単位）が定着しました。これらのパラメータに有意差は、女性と男性の間で見られませんでした。意義は、ダンの事後テストに続いて、ノンパラメトリッククラスカル-ウォリス一方向ANOVA検定（*、P≤0.05によって決定した; **、P≤0.01; ***はp≤0.001; ****、P </em&gt;の0.0001≤）。エラーバーは平均からの標準偏差を表します。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 4匹のマウス（雄2匹と雌2匹）は人道的などの非感染のコントロールを提供するために、感染前に、0日目に安楽死させ、剖検のために調製しました。また、各ケージから1マウスを人道的に安楽死させ、剖検のために準備日2に、4、7、14、攻撃後（ 図1A）。盲腸内容物中のクロストリジウム・ディフィシレの列挙は、各剖検で行いました。糞便列挙との一致では、何のクロストリジウム・ディフィシルは、前のC.ディフィシルでのチャレンジにマウスの盲腸内容物は検出されませんでした。 48時間チャレンジ後に、マウスは、盲腸内容物のグラムあたりのクロストリジウム・ディフィシルの約10 8 CFUのが定着した（ 図図2（a））。全てのマウスは、永続的に、実験を通して植民地化したままでした。 盲腸内容はまた、ベロ細胞毒性アッセイを用いてC.ディフィシル毒素の存在を感染を通して評価しました。 C.ディフィシル毒素の細胞毒性の証拠は、チャレンジ（ 図2B）の前に盲腸内容物中に検出されませんでした。 C.ディフィシル細胞毒性は、 クロストリジウム・ディフィシルの胞子で2日間のチャレンジ後に検出し、感染を通じて持続しました。 クロストリジウム・ディフィシルとかなり均一な植民地にもかかわらず、 クロストリジウム・ディフィシル細胞傷害活性のレベルの変化は個々のマウス（ 図2B）で明らかです。 Vero細胞の細胞毒性アッセイ中にクロストリジウム・ディフィシル抗毒素で中和マウス盲腸内容物の大部分および細胞毒性の証拠はなかった（ベロCE行っ希釈で）丸めてしまう。他の希釈で丸めVero細胞の証拠で、10 -1希釈（サンプルが最も集中している希釈）で100％の細胞毒性-しかし、いくつかの個々のマウスは、再試験にもかかわらず、50の証拠を持っていました。このアッセイで利用抗毒素はヤギ27で製造した、C。ディフィシル毒素に対する中和ポリクローナル抗体です。したがって、この抗毒素は菌株R20291によって生成されたC.ディフィシルバイナリ毒素を中和しないことがあります。しかし、C。ディフィシルバイナリ毒素は、10細胞傷害性であるとは考えられません。抗毒素またはC.ディフィシル毒素がこの発見に貢献することができた以外の他の細胞毒性剤の存在により中和することができない過度のC.ディフィシル毒素 。 80％Vero細胞の丸めと各サンプルの最後の希釈は持っていたので、この観察は、これらのサンプルについての細胞毒性力価の決意に影響を及ぼしませんでした指定された希釈率で抗毒素と組み合わせた細胞毒性の証拠はありません。 <img alt="図2" src="/files/ftp_upload/54850/54850fig2.jpg" /> 図2：Cでの感染時の盲腸および細胞毒性の植民地。ディフィシル R20291。 A）マウスの盲腸は永続的に臨床徴候の解像度および観察された体重増加にもかかわらず、実験を通してクロストリジウム・ディフィシルが定着したままでした。 2日後の課題で評価した場合、すべてのマウスは、盲腸内容物のグラム当たり10を超える8 CFUのが定着しました。 Cでの感染を通して盲腸内容物からB）ベロ細胞毒性アッセイ。ディフィシル R20291。 C.ディフィシレ胞子とチャレンジ後0日目に比べて2日目、4上のログ10盲腸内容物のグラムあたりの毒素の逆数希釈で測定されるように、マウスは、細胞毒性の有意なレベルを持っていた、と7攻撃後（中央値のrepresenteラインからD）。 **はp≤0.01; ***はp≤0.001; ****、P≤0.0001意義はダンの事後テスト（*、P≤0.05に続くノンパラメトリッククラスカル-ウォリス一方向ANOVA検定によって決定しました）。エラーバーは平均からの標準偏差を表します。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 組織学的評価は盲検法でボード認定の獣医病理学者によって行われることをお勧めします。組織のスコアリングのために、0-4の数値スコアリングシステムは、個別に、以前に公開されたスコアリングスキーム17に基づいて、回腸、盲腸および結腸に浮腫、炎症性細胞浸潤、及び上皮細胞の損傷を評価するために使用すべきです。 盲検病理組織学的検査時にC.ディフィシル R20291とネズミ回腸、盲腸および結腸次の挑戦のネーションは、最も重要な病理学的変化は盲腸（ 図3）で認められました。総盲腸組織学的スコアは、0日目と2日目と4攻撃後の間で有意に異なっていました。有意な病変は、感染を通して、回腸（ 図3）で認められませんでした。より軽度の病変は結腸において認められました。総結腸組織学的スコアは、0日目と2日目と7、チャレンジ後（ 図3）との間に有意差が認められました。 感染を通じて回腸、盲腸および結腸の代表的なH＆E切片は盲検ボード認定の獣医病理学者によって決定される各画像は、そのそれぞれの合計組織学的スコアおよび上皮損傷、炎症、および浮腫のための個々のスコアを持っている、図4で利用可能です（ SAM）。 Cでの感染の間にネズミ回腸、盲腸の組織学的スコアリング、およびコロン：図3。ディフィシル R20291。上皮損傷、炎症、および浮腫総組織学スコアを評価パラメータのすべての3つのスコアを加算することにより算出しました。有意な組織病理学的変化は、感染を通して回腸に認められませんでした。盲腸組織は、CDIの際に最も重要な組織学的病変を含んでいました。総盲腸組織学的スコアは、2日目と4攻撃後に0日目から有意に異なっていました。より軽度の病変は結腸において認められました。総結腸組織学的スコアは、2日目と7攻撃後に0日目から有意に異なっていました。意義は、ダンの事後テストに続いて、ノンパラメトリッククラスカル-ウォリス一方向ANOVA検定（*、P≤0.05によって決定した; **、P≤0.01; ***はp804; 0.001; ****、P≤0.0001）。エラーバーは平均からの標準偏差を表します。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/54850/54850fig4.jpg" /> 図4：Cでの感染の間に組織病理学。ディフィシル R20291。このパネルには、C。ディフィシル R20291での感染を通じて回腸、盲腸および結腸の代表的なH＆Eセクションが含まれています。総組織学的スコアは、対応する日と記載されている組織のための括弧（）内にある：回腸日0（0）、2日目（1）、4日（0）、7日目（0）、および14日目（0） ;盲腸日の0（0）、2日目（5）、4日目（4）、7日目（3）、及び14日目（3）。結腸日0（0）、2日目（4）、4日目（1）、7日目（4）、及び14日目（2）。顕微鏡写真は、光顕微鏡で得られました5メガピクセルのデジタルカメラとそれに付随するソフトウェアと電子（スケールバーは200μmで表します）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

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 ...

cefoperazone-treated-mouse-model-clinically-relevant-clostridium

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

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

12.2K ビュー.  North Carolina State University College of Veterinary Medicine. 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...

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

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

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

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

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

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

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

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

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 ...

Influenza A Virus Studies in a Mouse Model of Infection

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 considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using 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 ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

発注済み、緑膿菌ひずみ PA14 トランスポゾン挿入変異体の非冗長ライブラリのレプリケーション

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

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 ...

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

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

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

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

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

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

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

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 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

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

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

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, ...