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.

Declaración de ética: El Comité Institucional de Cuidado de Animales y el empleo (IACUC) en Carolina del Norte Facultad de Medicina Veterinaria de la Universidad Estatal (NCSU) aprobó este estudio. El Cuidado de Animales NCSU y usar la directiva aplica las normas y directrices establecidas en la Ley de Ley y Extensión Investigación en Salud Animal Welfare de 1985. Las instalaciones de los animales de laboratorio en la NCSU adherirse a las directrices establecidas en la Guía para el Cuidado y Uso de Animale...

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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 es un anaerobio,, Bacillus formador de esporas gram-positiva que causa diarrea que amenaza la vida 1. Infección por C. difficile (CDI) se asocia con una mayor morbilidad y mortalidad humana y los resultados en más de $ 4.8 mil millones en costos de salud por año 1-4. En 2013, los Centros para el Control y Prevención de Enfermedades clasifican C. difficile como un riesgo de resistencia a antibióticos urgente, lo que indica que presenta una amenaza inmediata para la salud pública 1. Actualmente, el tratamiento antibiótico con vancomicina y metronidazol se consideran el estándar de cuidado para CDI 5. Desafortunadamente, la recurrencia de CDI después del tratamiento exitoso con antibióticos es alta, se produce en 20 - 30% de los pacientes 2,5-7. Por lo tanto, el descubrimiento de nuevas terapias contra este patógeno entérico es necesario. Para evaluar la terapéutica frente a C. difficile, un modelo animal que se aproxima la enfermedad humana en corriente alternaSe necesita cepa linically relevantes. Inicialmente, se establecieron los postulados de Koch para C. difficile en 1977, utilizando un modelo de hámster sirio clindamicina tratados con 8. Este modelo todavía se utiliza hoy en día para investigar los efectos de toxinas de C. difficile en la patogénesis 9,10. Sin embargo, el CDI en el modelo de hámster se traduce en altas tasas de mortalidad y no se aproxima a las manifestaciones de la enfermedad insidiosa crónicas que se pueden ver en los seres humanos con CDI 10,11. Sobre la base de la disponibilidad y accesibilidad de reactivo de plataformas murinas en investigación, un modelo de ratón de CDI es relevante. En 2008, un modelo sólido ratón de CDI se estableció mediante el tratamiento de ratones con un cóctel de antibióticos en el agua potable (kanamicina, gentamicina, colistina, metronidazol y vancomicina) durante 3 días, seguido de una inyección intraperitoneal de clindamicina 12. Este ratones que son más susceptibles a la colitis CDI y severa. Dependering de la dosis de inóculo administrado, una amplia gama de signos clínicos y la letalidad se puede observar utilizando este modelo. Desde entonces, varios regímenes de antibióticos se han investigado que alteran la microbiota intestinal murino, disminuyendo la resistencia a la colonización hasta el punto en C. difficile puede colonizar el tracto gastrointestinal (revisado en Best et al. Y Lawley &amp; Young) 13,14. Más recientemente, un amplio espectro de cefalosporina, cefoperazona, dada en el agua de bebida durante 5 o 10 días reproducible hace ratones susceptibles a CDI 15. Puesto que la administración de cefalosporinas de tercera generación están asociados con un mayor riesgo de CDI en los seres humanos, el uso del modelo de cefoperazona refleja con mayor precisión la enfermedad 16 de origen natural. Cefoperazona tratados con ratones susceptibles a C. difficile han sido desafiados con las dos esporas de C. difficile y células vegetativas de una variedad de cepas que van de clínicarelevancia y la virulencia 17. A pesar de algunos de los estudios originales que utilizan C. difficile células vegetativas como la forma infecciosa, esporas de C. difficile se consideran el principal modo de transmisión 18. En la última década, C. difficile R20291, a / BI / 027 NAP1, ha surgido, causando epidemias de CDI 19,20. Hemos tratado de determinar la evolución clínica de la enfermedad, cuando los ratones tratados con cefoperazona fueron desafiados con la cepa de C. difficile clínicamente relevante y genéticamente manejable, R20291. Este protocolo se detalla el curso clínico, incluyendo pérdida de peso, la colonización bacteriana, la citotoxicidad de la toxina, y cambios histopatológicos en el tracto gastrointestinal de los ratones desafiados con esporas de C. difficile R20291. En general, este modelo de ratón demuestra ser una plataforma experimental valioso para CDI aproximación de la enfermedad humana. Este modelo de ratón caracterizado por lo tanto se puede utilizar para evaluar los efectosde nuevas terapias en la mejora de la enfermedad clínica y en la restauración de la resistencia a la colonización contra 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.

Durante un estudio representativo, 5 semanas de edad C57BL / 6 ratones WT fueron pretratados con cefoperazona en su agua potable (0,5 mg / ml) durante 5 días y se dejaron a 2 días a lavar con agua potable regular. Los ratones fueron retados con 10 5 esporas de C. difficile R20291 mediante sonda oral en el día 0 (Figura 1A). Los ratones se controlaron para la pérdida de peso y los signos clínicos (letargo, inapetencia, diarrea, y la postura encorv...

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

Este protocolo se describe el modelo de ratón cefoperazona de la infección por Clostridium difficile (CDI), utilizando una cepa clínicamente relevante y genéticamente manejable, R20291. Énfasis en el seguimiento clínico de la enfermedad, C. difficile enumeración bacteriana, la citotoxicidad de la toxina, y los cambios histopatológicos en todo CDI en un modelo de ratón se detalla en el protocolo.

Cefoperazona tratados modelo de ratón clínicamente relevante<em&gt; Clostridium difficile</em&gt; R20291 Strain

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

El cultivo y mantenimiento<em&gt; Clostridium difficile</em&gt; En un ambiente anaeróbico

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Investigación

Inmunología e Infección

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

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

Prevención de Estrés Térmico efectos adversos en ratas<em&gt; Bacillus subtilis</em&gt; Strain

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

Protocolo para Caracterizar los Cambios Morfológicos de<em&gt; Clostridium difficile</em&gt; En respuesta al tratamiento antibiótico

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

Estudios de Virus de influenza A en un modelo murino de infección

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

Un análisis de Microarray de proteínas para la determinación serológica de anticuerpos las respuestas siguientes infección por Clostridium difficile específica de antígeno

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

Una purificación y análisis de actividad In Vitro de una (p) ppGpp sintetasa de 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

Un modelo de ratón controlado para la Sepsis Neonatal polimicrobiana

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

Medición de la velocidad de propagación de pulsos, la distensión y la tensión en un modelo de ratón de aneurisma aórtico abdominal

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

Construcción de mutantes en el serotipo 1 Streptococcus pneumoniae cepa 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

Uveítis autoinmune experimental: un modelo de ratón inflamatorio intraocular

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