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>
	<li>Lessa, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+Clostridium+difficile+Infection+in+the+United+States.">Burden of Clostridium difficile Infection in the United States.</a> New England Journal of Medicine. 372, 825-834 (2015).</li><li>Gerding, D. N., Lessa, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+Clostridium+difficile+infection+inside+and+outside+health+care+institutions.">The epidemiology of Clostridium difficile infection inside and outside health care institutions.</a> Infect Dis Clin North Am. 29, 37-50 (2015).</li><li>Dubberke, E. R., Olsen, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+Clostridium+difficile+on+the+healthcare+system.">Burden of Clostridium difficile on the healthcare system.</a> Clin Infect Dis. 55, 88-92 (2012).</li><li>Kociolek, L. K., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakthroughs+in+the+treatment+and+prevention+of+Clostridium+difficile+infection.">Breakthroughs in the treatment and prevention of Clostridium difficile infection.</a> Nat Rev Gastroenterol Hepatol. , (2016).</li><li>Kelly, C. P., LaMont, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile--more+difficult+than+ever.">Clostridium difficile--more difficult than ever.</a> N Engl J Med. 359, 1932-1940 (2008).</li><li>Louie, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fidaxomicin+versus+vancomycin+for+Clostridium+difficile+infection.">Fidaxomicin versus vancomycin for Clostridium difficile infection.</a> N Engl J Med. 364, 422-431 (2011).</li><li>Bartlett, J. G., Onderdonk, A. B., Cisneros, R. L., Kasper, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clindamycin-associated+colitis+due+to+a+toxin-producing+species+of+Clostridium+in+hamsters.">Clindamycin-associated colitis due to a toxin-producing species of Clostridium in hamsters.</a> The Journal of infectious diseases. 136, 701-705 (1977).</li><li>Kelly, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+reproducibility+of+the+NAP1/B1/027+epidemic+strain+R20291+in+the+hamster+model+of+infection.">Improving the reproducibility of the NAP1/B1/027 epidemic strain R20291 in the hamster model of infection.</a> Anaerobe. , (2016).</li><li>Kuehne, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+toxin+A,+toxin+B,+and+CDT+in+virulence+of+an+epidemic+Clostridium+difficile+strain.">Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difficile strain.</a> The Journal of infectious diseases. 209, 83-86 (2014).</li><li>Bartlett, J. G., Onderdonk, A. B., Cisneros, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clindamycin-associated+colitis+in+hamsters:+protection+with+vancomycin.">Clindamycin-associated colitis in hamsters: protection with vancomycin.</a> Gastroenterology. 73, 772-776 (1977).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+Clostridium+difficile-associated+disease.">A mouse model of Clostridium difficile-associated disease.</a> Gastroenterology. 135, 1984-1992 (2008).</li><li>Lawley, T. D., Young, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+to+study+Clostridium+difficile+infection+and+transmission.">Murine models to study Clostridium difficile infection and transmission.</a> Anaerobe. 24, 94-97 (2013).</li><li>Best, E. L., Freeman, J., Wilcox, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+for+the+study+of+Clostridium+difficile+infection.">Models for the study of Clostridium difficile infection.</a> Gut Microbes. 3, 145-167 (2012).</li><li>Reeves, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interplay+between+microbiome+dynamics+and+pathogen+dynamics+in+a+murine+model+of+Clostridium+difficile+Infection.">The interplay between microbiome dynamics and pathogen dynamics in a murine model of Clostridium difficile Infection.</a> Gut Microbes. 2, 145-158 (2014).</li><li>Owens, R. C., Donskey, C. J., Gaynes, R. P., Loo, V. G., Muto, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial-associated+risk+factors+for+Clostridium+difficile+infection.">Antimicrobial-associated risk factors for Clostridium difficile infection.</a> Clin Infect Dis. 46, 19-31 (2008).</li><li>Theriot, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cefoperazone-treated+mice+as+an+experimental+platform+to+assess+differential+virulence+of+Clostridium+difficile+strains.">Cefoperazone-treated mice as an experimental platform to assess differential virulence of Clostridium difficile strains.</a> Gut Microbes. 2, 326-334 (2011).</li><li>Martin, J. S., Monaghan, T. M., Wilcox, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection:+epidemiology,+diagnosis+and+understanding+transmission.">Clostridium difficile infection: epidemiology, diagnosis and understanding transmission.</a> Nat Rev Gastroenterol Hepatol. , (2016).</li><li>Connor, J. R., Johnson, S., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection+caused+by+the+epidemic+BI/NAP1/027+strain.">Clostridium difficile infection caused by the epidemic BI/NAP1/027 strain.</a> Gastroenterology. 136, 1913-1924 (2009).</li><li>He, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+and+global+spread+of+epidemic+healthcare-associated+Clostridium+difficile.">Emergence and global spread of epidemic healthcare-associated Clostridium difficile.</a> Nat Genet. 45, 109-113 (2013).</li><li>Perez, J., Springthorpe, V. S., Sattar, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clospore:+a+liquid+medium+for+producing+high+titers+of+semi-purified+spores+of+Clostridium+difficile.">Clospore: a liquid medium for producing high titers of semi-purified spores of Clostridium difficile.</a> Journal of AOAC International. 94, 618-626 (2011).</li><li>Sorg, J. A., Dineen, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+maintenance+of+Clostridium+difficile.">Laboratory maintenance of Clostridium difficile.</a> Curr Protoc Microbiol. , (2009).</li><li>Edwards, A. N., Suarez, J. M., McBride, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+and+maintaining+Clostridium+difficile+in+an+anaerobic+environment.">Culturing and maintaining Clostridium difficile in an anaerobic environment.</a> Journal of visualized experiments : JoVE. , e50787 (2013).</li><li>George, W. L., Sutter, V. L., Citron, D., Finegold, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+and+differential+medium+for+isolation+of+Clostridium+difficile.">Selective and differential medium for isolation of Clostridium difficile.</a> J Clin Microbiol. 9, 214-219 (1979).</li><li>Knoblaugh, S., Randolph-Habecker, J., Rath, S., Dintzis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comparative Anatomy and Histology. , 15-40 (2012).</li><li>Ammerman, N. C., Beier-Sexton, M., Azad, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+maintenance+of+Vero+cell+lines.">Growth and maintenance of Vero cell lines.</a> Curr Protoc Microbiol. , (2008).</li><li>Theriot, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic-induced+shifts+in+the+mouse+gut+microbiome+and+metabolome+increase+susceptibility+to+Clostridium+difficile+infection.">Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection.</a> Nat Commun. 5, 3114 (2014).</li><li>Koenigsknecht, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+establishment+of+Clostridium+difficile+infection+in+the+murine+gastrointestinal+tract.">Dynamics and establishment of Clostridium difficile infection in the murine gastrointestinal tract.</a> Infect Immun. 83, 934-941 (2015).</li><li>Theriot, C., Bowman, A., Young, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic-Induced+Alterations+of+the+Gut+Microbiota+Alter+Secondary+Bile+Acid+Production+and+Allow+for+Clostridium+difficile+Spore+Germination+and+Outgrowth+in+the+Large+Intestine.">Antibiotic-Induced Alterations of the Gut Microbiota Alter Secondary Bile Acid Production and Allow for Clostridium difficile Spore Germination and Outgrowth in the Large Intestine.</a> mSphere. 1, 00045 (2016).</li><li>Xiao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+catalog+of+the+mouse+gut+metagenome.">A catalog of the mouse gut metagenome.</a> Nature biotechnology. 33, 1103-1108 (2015).</li><li>Leslie, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+and+toxin+production+by+Clostridium+difficile+within+human+intestinal+organoids+result+in+disruption+of+epithelial+paracellular+barrier+function.">Persistence and toxin production by Clostridium difficile within human intestinal organoids result in disruption of epithelial paracellular barrier function.</a> Infect Immun. 83, 138-145 (2015).</li><li>Stabler, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genome+and+phenotypic+analysis+of+Clostridium+difficile+027+strains+provides+insight+into+the+evolution+of+a+hypervirulent+bacterium.">Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium.</a> Genome Biol. 10, 102 (2009).</li><li>Valiente, E., Dawson, L. F., Cairns, M. D., Stabler, R. A., Wren, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+new+PCR+ribotypes+from+the+hypervirulent+Clostridium+difficile+027+lineage.">Emergence of new PCR ribotypes from the hypervirulent Clostridium difficile 027 lineage.</a> J Med Microbiol. 61, 49-56 (2012).</li></ol>

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 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。

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

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

Universidad de Cartagena UDEC

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

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

ザ<em&gt;シトロrodentium</em&gt;マウスモデル：感染性大腸炎に勉強病原体とホスト貢献

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

免疫・感染学

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

の4Dのマルチモダリティイメージング<em&gt;シトロバクターrodentium</emマウスにおける&gt;感染症

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

クロストリジオイド・ディフィシルの幼虫ゼブラフィッシュ感染モデルの開発

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

発生生物学

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

マウスモデルにおけるUTIとCAUTIの樹立とその性状

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

医学

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 ...

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. Meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject. In particular circumstances, it acquires an invasive ability to penetrate the mucosal barrier and invades the bloodstream causing septicaemia. In the latest case, fulminating sepsis could arise even without the consequent development of meningitis. Conversely, bacteria could poorly multiply in the bloodstream, cross the blood brain barrier, reach the central nervous system, leading to fulminant meningitis. The murine models of bacterial meningitis represent a useful tool to investigate the host-pathogen interactions and to analyze the pathogenetic mechanisms responsible for this lethal disease. Although, several experimental model systems have been evaluated over the last decades, none of these were able to reproduce the characteristic pathological events of meningococcal disease. In this experimental protocol, we describe a detailed procedure for the induction of meningococcal meningitis in a mouse model based on the intracisternal inoculation of bacteria. The peculiar signs of human meningitis were recorded in the murine host through the assessment of clinical parameters (e.g., temperature, body weight), evaluation of survival rate, microbiological analysis and histological examination of brain injury. When using intracisternal (i.cist.) inoculum, meningococci complete delivery directly into cisterna magna, leading to a very efficient meningococcal replication in the brain tissue. A 1,000-fold increase of viable count of bacteria is observed in about 18 h. Moreover, meningococci are also found in the spleen, and liver of infected mice, suggesting that the liver may represent a target organ for meningococcal replication.

Here, we describe a method to induce meningococcal meningitis through an intracisternal route of infection in adult mice. We present a step by step protocol of meningococcal infection from the preparation of inoculum to the intracisternal infection; then record the animal survival and evaluate the bacterial loads in murine tissues.

インティリカレルナルデリバリーによるマウスにおける髄膜炎球炎血清群Cの誘導

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

マウスの遠位人工肛門、転用 c57bl/6 マウス大腸炎の新たなモデル

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

マウスにおけるガードネレラ膣膀胱曝露によって引き起こされる再発性大腸菌尿路感染症

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted considerable attention. Accumulating evidence suggests that dysbiotic microbiota plays an active role in triggering or worsening the inflammatory process in IBD and that fecal microbiota transplantation (FMT) is an attractive therapeutic strategy since transferring a healthy microbiota to IBD patient could restore the appropriate host-microbiota communication. However, the molecular mechanisms are unclear, and the efficacy of FMT has not been very well established. Thus, further studies in animal models of IBD are necessary. In this method, we applied FMT from wild-type C57BL/6J mice to IL-10 deficient mice, a widely used mouse model of colitis. The study elaborates on collecting fecal pellets from the donor mice, making the fecal solution/suspension, administering the fecal solution, and monitoring the disease. We found that FMT significantly mitigated the cardiac impairment in IL-10 knockout mice, underlining its therapeutic potential for IBD management.

The interaction of genetic susceptibility, mucosal immunity, and intestinal microecological environment is involved in the pathogenesis of inflammatory bowel disease (IBD). In this study, we applied fecal microbiota transplantation to IL-10 deficient mice and investigated its impact on colonic inflammation and heart function.

インターロイキン10欠損マウスモデルにおける糞便微生物叢移植の治療的評価

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted ...

カンジダ・アルビカンス関連カテーテル関連感染症のマウスモデルの確立

A Catheter-Related Candida albicans Infection Model in Mouse

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed on the outer surface of the catheter and lead to disseminated infections, which are fatal to patients. There are no effective prevention and treatment management in clinics. Therefore, it is urgent to establish an animal model of CRI for the preclinical screening of new strategies for its prevention and treatment. In this study, a polyethylene catheter, a widely used medical catheter, was inserted into the back of the BALB/c mice after hair removal. Candida albicans ATCC MYA-2876 (SC5314) expressing enhanced green fluorescent protein was subsequently inoculated on the skin's surface along the catheter. Intense fluorescence was observed on the surface of the catheter under a fluorescent microscope 3 days later. Mature and thick biofilms were found on the surface of the catheter via scanning electron microscopy. These results indicated the adhesion, colonization, and biofilm formation of candida albicans on the surface of the catheter. The hyperplasia of the epidermis and the infiltration of inflammatory cells in the skin specimens indicated the histopathological changes of the CRI-associated skin. To sum up, a mouse CRI model was successfully established. This model is expected to be helpful in the research and development of therapeutic management for candida albicans associated CRI.

著者スポットライト:蛍光タンパク質標識を使用したカテーテル感染症におけるカンジダ・アルビカンス検出の強化 

We establish a mouse model of C.albicans-associated catheter-related infection (CRI), in which biofilm forms on the catheter, and the interaction between C.albicans and host correlates well with the clinical CRI. This model helps screen therapies for C.albicans biofilm-associated CRI, laying a foundation for clinical transformation.

マウスにおけるカテーテル関連 カンジダ・アルビカンス 感染モデル

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed ...

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

要約

さらに動画を探す

この動画の章

ザ

の4Dのマルチモダリティイメージング

クロストリジオイド・ディフィシルの幼虫ゼブラフィッシュ感染モデルの開発

マウスモデルにおけるUTIとCAUTIの樹立とその性状

新生児モデル敗血症マウスの制御モデル

インティリカレルナルデリバリーによるマウスにおける髄膜炎球炎血清群Cの誘導

マウスの遠位人工肛門、転用 c57bl/6 マウス大腸炎の新たなモデル

マウスにおけるガードネレラ膣膀胱曝露によって引き起こされる再発性大腸菌尿路感染症

インターロイキン10欠損マウスモデルにおける糞便微生物叢移植の治療的評価

マウスにおけるカテーテル関連カンジダ・アルビカンス感染モデル

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

要約

さらに動画を探す

この動画の章

ザ

の4Dのマルチモダリティイメージング

クロストリジオイド・ディフィシルの幼虫ゼブラフィッシュ感染モデルの開発

マウスモデルにおけるUTIとCAUTIの樹立とその性状

新生児モデル敗血症マウスの制御モデル

インティリカレルナルデリバリーによるマウスにおける髄膜炎球炎血清群Cの誘導

マウスの遠位人工肛門、転用 c57bl/6 マウス大腸炎の新たなモデル

マウスにおけるガードネレラ膣膀胱曝露によって引き起こされる再発性大腸菌尿路感染症

インターロイキン10欠損マウスモデルにおける糞便微生物叢移植の治療的評価

マウスにおけるカテーテル関連 カンジダ・アルビカンス 感染モデル

マウスにおけるカテーテル関連カンジダ・アルビカンス感染モデル