-Cefoperazone behandelde muismodel van klinisch relevante<em> Clostridium difficile</em> 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 de standaard van zorg voor CDI 5. Helaas, herhaling van CDI na een succesvolle behandeling met antibiotica is hoog, optreedt in 20-30% van de patiënten 2,5-7. Daarom is de ontdekking van nieuwe therapieën tegen deze darmpathogeen noodzakelijk. Om therapieën te onderzoeken tegen C. difficile, een diermodel benadert de ziekte bij de mens in aclinically relevante stam nodig is.
Aanvankelijk werden postulaten vastgesteld voor C. difficile in 1977 met een clindamycine behandeld Syrische hamster model 8. Dit model wordt nog steeds gebruikt om de effecten van C. difficile toxinen onderzoeken naar pathogenese 9,10. Echter, CDI in het hamstermodel resulteert in hoge sterfte en niet nader chronische sluipende ziektemanifestaties die kunnen worden gezien bij mensen met CDI 10,11. Gebaseerd op de toegankelijkheid en reagens beschikbaarheid van murine platforms in onderzoek, een muismodel van CDI relevant.
In 2008, een robuust muismodel van CDI werd opgericht door de behandeling van muizen met een antibioticum cocktail in drinkwater (kanamycine, gentamicine, colistine, metronidazol en vancomycine) gedurende 3 dagen, gevolgd door een intraperitoneale injectie van clindamycine 12. Dit teruggegeven muizen gevoelig voor CDI en ernstige colitis. afhangening op de entstof dosis toegediend, kan een reeks klinische symptomen en sterfte worden geobserveerd met behulp van dit model. Sindsdien zijn diverse antibiotische regimes onderzocht dat de murine darmflora verandert, aflopend kolonisatieweerstand tot het punt waar C. difficile het maagdarmkanaal kan koloniseren (beschreven door Best et al. En Lawley & Young) 13,14.
Meer recent is een breed spectrum cefalosporine, cefoperazon, gezien in het drinkwater gedurende 5 of 10 dagen maakt reproduceerbaar muizen gevoelig voor CDI 15. Aangezien toediening van de derde generatie cefalosporinen geassocieerd met een verhoogd risico van CDI bij mensen gebruik van de cefoperazon model beter weergeeft nature voorkomende ziekte 16. -Cefoperazone behandelde muizen gevoelig voor C. difficile werden uitgedaagd met zowel C. difficile sporen en vegetatieve cellen van verschillende stammen variërend in klinischerelevantie en virulentie 17. Ondanks dat een aantal van de oorspronkelijke studies gebruik te maken van C. difficile vegetatieve cellen als de besmettelijke vorm, C. difficile sporen beschouwd als de belangrijkste wijze van overdracht 18.
In het laatste decennium, C. difficile R20291, een NAP1 / BI / 027 stam, is ontstaan, waardoor epidemieën van CDI 19,20. Wij hebben geprobeerd om het klinisch beloop van de ziekte te bepalen wanneer-cefoperazon behandelde muizen werden uitgedaagd met de klinisch relevante en genetisch handelbaar C. difficile stam, R20291. Dit protocol geeft de klinisch verloop, zoals gewichtsverlies, bacteriële kolonisatie, toxine cytotoxiciteit en histopathologische veranderingen in het maagdarmkanaal van muizen geprikkeld met C. difficile R20291 sporen. Kortom, dit muismodel blijkt een waardevolle experimenteel platform voor CDI benaderen van ziekten bij de mens zijn. Dit kenmerk muismodel kan dus worden gebruikt om de effecten te evaluerenvan nieuwe therapieën voor de verbetering van de klinische ziekte en het herstel van kolonisatieweerstand tegen C. difficile.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| #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 37C 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 37C prior to use |
| 1ml 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 37C 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 |
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