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.

Etiska riktlinjer: Institutional Animal Care och användning kommittén (IACUC) vid North Carolina State University College of Veterinary Medicine (NCSU) godkänt denna studie. NCSU Animal Care och använda policy gäller normer och riktlinjer som anges i djurskyddslagen och hälsoforskning Extension Act från 1985. Laboratoriedjuranläggningar vid NCSU följer riktlinjer som anges i handledningen för vård och användning av försöksdjur. Djurens hälsostatus bedömdes dagligen, och döende djur humant av...

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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...

Clostridium difficile är en anaerob, grampositiv, sporbildande bakterie som orsakar livshotande diarré 1. C. difficile-infektion (CDI) är associerad med ökad mänsklig morbiditet och mortalitet och resulterar i mer än $ 4,8 miljarder sjukvårdskostnader per år 1-4. 2013, de Centers for Disease Control and Prevention kategoriseras C. difficile som en brådskande antibiotikaresistens risk, vilket indikerar att det utgör ett akut hot mot folkhälsan en. För närvarande är antibiotikabehandling med vankomycin och metronidazol anses normen av omsorg för CDI 5. Olyckligtvis är återkommande CDI efter framgångsrik behandling med antibiotika hög, förekommer i 20-30% av patienterna 2,5-7. Därför är det nödvändigt att upptäckten av nya läkemedel mot denna enter patogen. För att utvärdera terapeutika mot C. difficile, en djurmodell som approximerar den mänskliga sjukdomen i acbehövs linically relevanta stammen. Inledningsvis var Kochs postulat fastställts för C. difficile 1977 med hjälp av en klindamycin behandlad syrisk hamster modell 8. Denna modell är fortfarande används idag för att undersöka effekterna av C. difficile-toxiner på patogenes 9,10. Men CDI i hamstermodellen resulterar i hög dödlighet och inte närma kroniska lömsk sjukdom manifestationer som kan ses hos människor med CDI 10,11. Baserad på tillgänglighet och reagens tillgängligheten av murina plattformar inom forskning, är en musmodell av CDI relevanta. Under 2008 genomfördes en robust musmodell av CDI fastställts genom att behandla möss med ett antibiotikum cocktail i dricksvatten (kanamycin, gentamicin, kolistin, metronidazol, och vankomycin) under 3 dagar följt av en intraperitoneal injektion av klindamycin 12. Detta renderade möss mottagliga för CDI och svår kolit. Beroing på ympen administrerade dosen, kan observeras en rad kliniska tecken och dödlighet med användning av denna modell. Sedan denna tid har olika antibiotiska regimer undersökts som förändrar den murina tarmfloran, minskande kolonisering motstånd mot den punkt där C. difficile kan kolonisera mag-tarmkanalen (översikt i Best et al., Och Lawley &amp; Young) 13,14. På senare tid har ett brett spektrum cefalosporin, cefoperazon, ges i dricksvattnet i 5 eller 10 dagar gör reproducerbart möss mottagliga för CDI 15. Eftersom administrering av tredje generationens cefalosporiner är förknippade med en ökad risk för CDI hos människor, användning av cefoperazon modellen bättre speglar naturligt förekommande sjukdomar 16. Cefoperazon-behandlade möss mottagliga för C. difficile har ifrågasatts med både C. difficile sporer och vegetativa celler av olika stammar som sträcker sig i kliniskrelevans och virulens 17. Trots vissa av de ursprungliga studier där man använt C. difficile vegetativa celler som smitt form, C. difficile sporer anses vara den stora överföringsteknik 18. Under det senaste decenniet, C. difficile R20291, en NAP1 / BI / 027-stammen, har dykt upp, vilket epidemier av CDI 19,20. Vi försökte bestämma den kliniska sjukdomsförloppet när cefoperazon behandlade mössen med kliniskt relevant och genetiskt foglig C. difficile stam, R20291. Detta protokoll detaljer det kliniska förloppet, inklusive viktförlust, bakteriell kolonisering, toxin cytotoxicitet, och histopatologiska förändringar i mag-tarmkanalen hos möss utmanade med C. difficile R20291 sporer. Sammantaget visar denna musmodell för att vara en värdefull experimentell plattform för CDI tillnärmning mänskliga sjukdomar. Denna kännetecknas musmodell kan därför användas för att bedöma effekternaav nya läkemedel på förbättring av klinisk sjukdom och om återställande av kolonisationen motstånd mot C. difficile.

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	<tbody>
		<tr height="21">
			<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>
		<tr height="21">
			<td height="21" style="height:21px;">1% Peniciilin/Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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.

Under en representativ studie var 5 veckor gamla C57BL / 6 WT möss förbehandlade med cefoperazon i sitt dricksvatten (0,5 mg / ml) under 5 dagar och fick en 2-dagars tvätta ur med regelbunden dricksvatten. Möss utmanades med 10 5 sporer av C. difficile R20291 via oral sondmatning på dag 0 (Figur 1A). Möss övervakades för viktminskning och kliniska tecken (letargi, aptitlöshet, diarré, och krökt kroppshållning) av CDI under 14 dagar. Utmani...

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

Detta protokoll beskriver cefoperazon musmodell av Clostridium difficile-infektion (CDI) med en kliniskt relevant och genetiskt foglig stam, R20291. Betoning på klinisk övervakning sjukdom, C. difficile bakteriell uppräkning, toxin cytotoxicitet och histopatologiska förändringar under CDI i en musmodell beskrivs i protokollet.

Cefoperazon behandlade musmodell av kliniskt relevanta<em&gt; Clostridium difficile</em&gt; Stam 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

Odling och underhålla<em&gt; Clostridium difficile</em&gt; I en anaerob miljö

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

Förebyggande av värmestress Biverkningar hos råttor efter<em&gt; Bacillus subtilis</em&gt; Stam

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

Ett protokoll för att karaktärisera de morfologiska förändringarna av<em&gt; Clostridium difficile</em&gt; Som svar på antibiotisk behandling

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

Influensa A Virus studier på en musmodell av infektion

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

En Protein Microarray analys för serologisk bestämning av Antigen-specifika antikroppen Svaren följande Clostridium difficile infektion

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

En rening och In Vitro aktivitet Assay för en (p) ppGpp kväveoxidsyntas från Clostridium difficile

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

En kontrollerad musmodell för Neonatal polymikrobiella Sepsis

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

Mätning av pulsförökningshastighet, distensibilitet och stam i en bukkolorektal aneurysm musmodell

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

Konstruera mutanter i Serotyp 1 Streptococcus pneumoniae stam 519/43

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

Experimentell autoimmun uveit: En intraokulär inflammatorisk musmodell

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