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.

नैतिक कथन: संस्थागत पशु की देखभाल और उपयोग समिति (IACUC) उत्तरी कैरोलिना स्टेट यूनिवर्सिटी कॉलेज पशु चिकित्सा में (NCSU) इस अध्ययन को मंजूरी दे दी। NCSU पशु की देखभाल और नीति NCSU पर 1985 प्रयोगशाला पशु सुविधाओं के प?...

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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...

क्लोस्ट्रीडियम बेलगाम एक anaerobic, ग्राम पॉजिटिव, बीजाणु के गठन बेसिलस कि जीवन के लिए खतरा दस्त 1 का कारण बनता है। सी बेलगाम संक्रमण (सीडीआई) प्रति वर्ष 1-4 स्वास्थ्य देखभाल की लागत में अधिक 4.8 अरब $ में वृद्धि हुई मानव रुग्णता और मृत्यु दर और परिणामों के साथ जुड़ा हुआ है। 2013 में, रोग नियंत्रण और रोकथाम के लिए केंद्र एक जरूरी एंटीबायोटिक प्रतिरोध जोखिम के रूप में सी बेलगाम वर्गीकृत, यह दर्शाता है कि यह सार्वजनिक स्वास्थ्य 1 के लिए एक तत्काल खतरा बन गया है। वर्तमान में, vancomycin के साथ एंटीबायोटिक उपचार और metronidazole CDI 5 के लिए देखभाल के मानक माना जाता है। रोगियों 2,5-7 का 30% - दुर्भाग्य से, एंटीबायोटिक दवाओं के साथ सफल इलाज के बाद CDI की पुनरावृत्ति 20 में होने वाली उच्च है। इसलिए, इस आंतों का रोगज़नक़ के खिलाफ उपन्यास चिकित्सा की खोज के लिए आवश्यक है। सी बेलगाम, एक पशु मॉडल एसी में मानव रोग का अनुमान करने के खिलाफ चिकित्सा विज्ञान का मूल्यांकन करने के लिएlinically प्रासंगिक तनाव की जरूरत है। प्रारंभ में, कोच की अभिधारणा 1977 में सी बेलगाम के लिए स्थापित किए गए थे एक clindamycin इलाज सीरियाई हम्सटर मॉडल 8 का उपयोग। यह मॉडल आज भी उपयोग किया जाता है रोगजनन 9,10 पर सी बेलगाम विषाक्त पदार्थों के प्रभाव की जांच करने के लिए। हालांकि, CDI हम्सटर मॉडल में उच्च मृत्यु दर में यह परिणाम है और पुरानी घातक बीमारी अभिव्यक्तियों CDI 10,11 के साथ मनुष्य में देखा जा सकता है कि लगभग नहीं है। अनुसंधान के क्षेत्र में murine प्लेटफार्मों की पहुंच और अभिकर्मक उपलब्धता के आधार पर, CDI के एक माउस मॉडल प्रासंगिक है। 2008 में, CDI के एक मजबूत माउस मॉडल 3 दिन clindamycin 12 की intraperitoneal इंजेक्शन द्वारा पीछा के लिए पीने के पानी में एक एंटीबायोटिक कॉकटेल (केनामाइसिन, जेंटामाइसिन, colistin, metronidazole, और vancomycin) के साथ चूहों के इलाज से स्थापित किया गया था। यह प्रदान की गई चूहों CDI और गंभीर कोलाइटिस के लिए अतिसंवेदनशील। निर्भर करती हैinoculum खुराक प्रशासित पर हैैं, नैदानिक ​​लक्षण और मारक की एक सीमा के इस मॉडल का उपयोग कर देखा जा सकता है। इस समय के बाद से, विभिन्न एंटीबायोटिक परहेजों 13,14 जांच की गई है कि murine पेट microbiota परिवर्तन, इस मुद्दे पर जहां सी बेलगाम जठरांत्र पथ उपनिवेश स्थापित कर सकते हैं बसाना प्रतिरोध कम (लॉली एंड यंग बेस्ट एट अल में समीक्षा की। और)। हाल ही में, एक व्यापक स्पेक्ट्रम cephalosporin, Cefoperazone, 5 या 10 दिनों के लिए पीने के पानी में दिए गए reproducibly CDI 15 को अतिसंवेदनशील चूहों प्रदान करता है। चूंकि तीसरी पीढ़ी सेफालोस्पोरिन्स के प्रशासन मानव में CDI का एक बढ़ा जोखिम के साथ जुड़े रहे हैं, Cefoperazone मॉडल के उपयोग को और अधिक सही स्वाभाविक रूप से होने वाली बीमारी 16 को दर्शाता है। Cefoperazone इलाज चूहों सी बेलगाम के लिए अतिसंवेदनशील दोनों सी बेलगाम बीजाणुओं और नैदानिक में लेकर उपभेदों की एक किस्म की वनस्पति कोशिकाओं के साथ चुनौती दी गई हैप्रासंगिकता और डाह 17। संक्रामक फार्म के रूप में सी बेलगाम वनस्पति कोशिकाओं के उपयोग के मूल अध्ययन से कुछ के बावजूद, सी बेलगाम बीजाणुओं संचरण 18 वर्ष की प्रमुख विधा माना जाता है। पिछले दशक में, सी बेलगाम R20291, एक NAP1 / बीआई / 027 तनाव, उभरा है, CDI 19,20 की महामारी के कारण। हम रोग के नैदानिक पाठ्यक्रम निर्धारित करने के लिए जब Cefoperazone इलाज चूहों नैदानिक प्रासंगिक और आनुवंशिक रूप से विनयशील सी बेलगाम तनाव, R20291 साथ चुनौती दी थी की मांग की। इस प्रोटोकॉल नैदानिक पाठ्यक्रम विवरण, वजन घटाने, बैक्टीरियल उपनिवेशवाद, विष cytotoxicity, और सी बेलगाम R20291 बीजाणुओं के साथ चुनौती दी चूहों के जठरांत्र संबंधी मार्ग में परिवर्तन सहित histopathological। कुल मिलाकर, इस माउस मॉडल CDI मानव रोग का अनुमान करने के लिए एक मूल्यवान प्रयोगात्मक मंच साबित होता है। यह विशेषता माउस मॉडल इस प्रकार प्रभाव का आकलन करने के लिए उपयोग किया जा सकतानैदानिक रोग के सुधार पर और सी बेलगाम के खिलाफ उपनिवेशन प्रतिरोध की बहाली पर उपन्यास चिकित्सा की।

<table border="0" cellpadding="0" cellspacing="0" width="1037">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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.

एक प्रतिनिधि अध्ययन के दौरान, 5 सप्ताह पुराने C57BL / 6 WT चूहों 5 दिनों के लिए उनके पीने के पानी (0.5 मिलीग्राम / एमएल) में Cefoperazone साथ pretreated और एक 2 दिन नियमित रूप से पीने के पानी के साथ बाहर धोने की अनुमति दी ...

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

इस प्रोटोकॉल क्लोस्ट्रीडियम बेलगाम संक्रमण (सीडीआई) एक चिकित्सकीय प्रासंगिक और आनुवंशिक रूप से विनयशील तनाव, R20291 का उपयोग करने का Cefoperazone माउस मॉडल की रूपरेखा। नैदानिक रोग निगरानी, सी बेलगाम बैक्टीरियल गणन, विष cytotoxicity, और एक माउस मॉडल में CDI भर histopathological परिवर्तन पर जोर प्रोटोकॉल में विस्तृत रहे हैं।

Cefoperazone इलाज के माउस मॉडल नैदानिक ​​प्रासंगिक<em&gt; क्लोस्ट्रीडियम बेलगाम</em&gt; तनाव 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

संवर्धन और अनुरक्षण<em&gt; क्लोस्ट्रीडियम बेलगाम</em&gt; एक anaerobic वातावरण में

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

गर्मी की रोकथाम से चूहे में प्रतिकूल प्रभाव तनाव<em&gt; बेसिलस subtilis</em&gt; तनाव

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

एक प्रोटोकॉल का आकृति परिवर्तन करने के लिए<em&gt; क्लॉस्ट्रिडियम डिफ़िज़ील</em&gt; एंटीबायोटिक उपचार के जवाब में

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

इंफ्लूएंजा संक्रमण के एक माउस मॉडल में एक वायरस अध्ययन

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

क्लोस्ट्रीडियम डिफीसाइल संक्रमण के बाद प्रतिजन-विशिष्ट एंटीबॉडी प्रतिक्रियाओं के सीरम वैज्ञानिक निर्धारण के लिए एक प्रोटीन Microarray परख

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

एक शुद्धि और इन विट्रो में गतिविधि परख के लिए एक (पी) ppGpp Synthetase से क्लोस्ट्रीडियम डिफीसाइल

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

नवजात Polymicrobial पूति के लिए एक नियंत्रित माउस मॉडल

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

पेट महाधमनी एन्यूरिज्म माउस मॉडल में पल्स प्रचार वेग, डिस्टेन्सिबिलिटी और तनाव का मापन

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

सीरोटाइप 1 स्ट्रेप्टोकोकस निमोनिया स्ट्रेन 519/43 में उत्परिवर्ती का निर्माण

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

प्रायोगिक ऑटोम्यून्यून यूवाइटिस: एक इंट्राओकुलर भड़काऊ माउस मॉडल

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

Cefoperazone इलाज के माउस मॉडल नैदानिक ​​प्रासंगिक

Cefoperazone इलाज के माउस मॉडल नैदानिक प्रासंगिक