-Cefoperazone behandelde muismodel van klinisch relevante Clostridium difficile Strain R20291

Summary

Dit protocol beschrijft de cefoperazon muismodel van Clostridium difficile infectie (CDI) met een klinisch relevante en genetisch handelbaar stam, R20291. Nadruk op klinische ziekte monitoring, C. difficile bacteriële opsomming, toxine cytotoxiciteit en histopathologische veranderingen in heel CDI in een muismodel worden beschreven in het protocol.

Abstract

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.

Introduction

Clostridium difficile is een anaërobe, grampositieve, sporenvormende bacil die levensbedreigende diarree ¹ veroorzaakt. C. difficile infectie (CDI) is geassocieerd met een verhoogde menselijke morbiditeit en mortaliteit en resulteert in meer dan $ 4800000000 in de kosten van de gezondheidszorg per jaar ^1-4. In 2013, de Centers for Disease Control and Prevention gecategoriseerd C. difficile als een dringende antibioticaresistentie risico, wat aangeeft dat het een dringende bedreiging voor de volksgezondheid ¹ vormt. Op dit moment, antibiotische behandeling met vancomycine en metronidazol worden beschouwd als ....

Protocol

Ethische verklaring:
De Institutional Animal Care en gebruik Comite (IACUC) aan de North Carolina State University College of Veterinary Medicine (NCSU) goedgekeurd deze studie. De NCSU Animal Care en gebruik beleid geldt normen en richtlijnen uiteengezet in de Animal Welfare Act en Health Research Extension Act van 1985. proefdierfaciliteiten bij NCSU voldoen aan richtlijnen uiteengezet in de Gids voor de zorg en het gebruik van proefdieren. De dieren 'gezondheidsstatus werden dagelijks geëvalueerd en stervende dieren werden op humane wijze gedood door CO ₂ stikken, gevolgd door secundaire maatregelen. Opgeleide technici dier of een dieren....

Results

Gedurende een representatieve studie werden 5 weken oude C57BL / 6-muizen voorbehandeld met WT cefoperazon in hun drinkwater (0,5 mg / ml) gedurende 5 dagen en men liet 2 dagen spoelen regelmatig drinkwater. Muizen werden geprikkeld met 10 ⁵ sporen van C. difficile R20291 via orale sondevoeding op dag 0 (Figuur 1A). De muizen werden gevolgd voor gewichtsverlies en klinische symptomen (lusteloosheid, gebrek aan eetlust, diarree en gebogen houding) van .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#62 Perisept Sporidicial Disinfectant Cleaner	SSS Navigator	48027	This product will require dilution as recommended by the manufacturer
0.22 μm filter	Fisherbrand	09-720-3	Alternative to filter plate for indivdiual samples tested in the Vero Cell Assay
0.25% Trypsin-EDTA	Gibco	25200-056	Needs to be heated in water bath at 37 °C prior to use
0.4% Trypan Blue	Gibco	15250-061
1% Peniciilin/Streptomycin	Gibco	15070-063
10% heat inactivated FBS	Gibco	16140-071	Needs to be heated in water bath at 37 °C prior to use
1 ml plastic syringe	BD Medical Supplies	309628
1x PBS	Gibco	10010-023
2 ml Micro Centrifuge Screw Cap	Corning	430917
96 well cell culture flat bottom plate	Costar Corning	CL3595
96 well filter plate	Millipore	MSGVS2210
Adhesive Seal	ThermoScientific	AB-0558
Bacto Agar	Becton Dickinson	214010	Part of TCCFA plates (see below)
Bacto Proteose Peptone	Becton Dickinson	211684	Part of TCCFA plates (see below)
Cefoperazone	MP Bioworks	199695
Cefoxitine	Sigma	C47856	Part of TCCFA plates (see below)
Clostridium difficile Antitoxin Kit	Tech Labs	T5000	Used as control for Vero Cell Assay
Clostridium difficile Toxin A	List Biological Labs	152C	Positive control for Vero Cell Assay
D-cycloserine	Sigma	C6880	Part of TCCFA plates (see below)
Distilled Water	Gibco	15230
DMEM 1x Media	Gibco	11965-092	Needs to be heated in water bath at 37 °C prior to use
Fructose	Fisher	L95500	Part of TCCFA plates (see below)
Hemocytometer	Bright-Line, Sigma	Z359629
KH2PO4	Fisher	P285-500	Part of TCCFA plates (see below)
MgSO4 (anhydrous)	Sigma	M2643	Part of TCCFA plates (see below)
Millex-GS 0.22 μm filter	Millex-GS	SLGS033SS	Filter for TCCFA plates
Na2HPO4	Sigma	S-0876	Part of TCCFA plates (see below)
NaCl	Fisher	S640-3	Part of TCCFA plates (see below)
Number 10 disposable scalpel blade	Miltex, Inc	4-410
PCR Plates	Fisherbrand	14230244
Plastic petri dish	Kord-Valmark Brand	2900
Sterile plastic L-shaped cell spreader	Fisherbrand	14-665-230
Syringe Stepper	Dymax Corporation	T15469
Taurocholate	Sigma	T4009	Part of TCCFA plates (see below)
Ultrapure distilled water	Invitrogen	10977-015
C57BL/6J Mice	The Jackson Laboratory	664	Mice should be 5 - 8 weeks of age
Olympus BX43F light microscope	Olympus Life Science
DP27 camera	Olympus Life Science
cellSens Dimension software	Olympus Life Science