Cefoperazon behandlade musmodell av kliniskt relevanta<em> Clostridium difficile</em> Stam R20291
Summary
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.
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 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 & 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.
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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