Denne protokol beskriver cefoperazon musemodel for Clostridium difficile infektion (CDI) ved anvendelse af en klinisk relevant og genetisk-tractable stamme, R20291. Fokus på klinisk sygdom overvågning, C. difficile bakteriel optælling, toksin cytotoksicitet, og histopatologiske ændringer i hele CDI i en musemodel er beskrevet i protokollen.
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.
Clostridium difficile er en anaerob, grampositiv, sporedannende bacillus, der forårsager livstruende diarré 1. C. difficile infektion (CDI) er forbundet med øget menneskelig sygelighed og dødelighed og resulterer i over $ 4.8 milliarder i udgifter til sundhedsvæsenet om året 1-4. I 2013, Centers for Disease Control og Forebyggelse kategoriseret C. difficile som et presserende antibiotikaresistens risiko, hvilket indikerer, at det udgør et presserende trussel mod den offentlige sundhed en. I øjeblikket er antibiotisk behandling med vancomycin og metronidazol overvejet standard for pleje af CDI 5. Desværre fornyet CDI efter vellykket behandling med antibiotika er høj, forekommer i 20 – 30% af patienterne 2,5-7. Derfor er det nødvendigt opdagelsen af nye lægemidler mod denne enterisk patogen. For at evaluere terapeutiske midler mod C. difficile, en dyremodel tilnærme den humane sygdom i acDer er behov linically-relevant stamme.
Indledningsvis blev Kochs postulater fastsat for C. difficile i 1977 under anvendelse af en clindamycin-behandlede syriske hamster model 8. Denne model er stadig udnyttes i dag til at undersøge virkningerne af C. difficile-toksiner på patogenese 9,10. Imidlertid CDI i hamstermodellen resulterer i høj dødelighed og ikke indbyrdes kroniske snigende sygdomsmanifestationer, der kan ses hos mennesker med CDI 10,11. Baseret på tilgængelighed og reagens tilgængeligheden af murine platforme i forskning, en musemodel for CDI er relevant.
I 2008 blev en robust musemodel af CDI etableret ved at behandle mus med et antibiotikum cocktail i drikkevand (kanamycin, gentamicin, colistin, metronidazol, og vancomycin) i 3 dage efterfulgt af en intraperitoneal injektion af clindamycin 12. Dette gøres mus følsomme over for CDI og alvorlig colitis. Afhænge afgigt af inokulum dosis indgivet, kan en række kliniske symptomer og letalitet observeres ved hjælp af denne model. Siden den tid har forskellige antibiotiske regimener blevet undersøgt som ændrer murine tarmens mikrobiota, faldende kolonisering modstand mod det punkt, hvor C. difficile kan kolonisere mavetarmkanalen (gennemgået i Best et al. Og Lawley & Young) 13,14.
For nylig en bredspektret cephalosporin, cefoperazon, givet i drikkevandet i 5 eller 10 dage reproducerbart gør mus modtagelige for CDI 15. Da administration af tredje-generations cephalosporiner er forbundet med en øget risiko for CDI i mennesker, anvendelse af cefoperazon model mere præcist afspejler naturligt forekommende sygdom 16. Cefoperazon-behandlede mus modtagelige for C. difficile er blevet udfordret med både C. difficile sporer og vegetative celler af forskellige stammer, der spænder i kliniskrelevans og virulens 17. Trods nogle af de oprindelige forsøg med C. difficile vegetative celler som den smitsomme form er C. difficile sporer betragtet den store smittemåde 18.
I det sidste årti, C. difficile R20291, en NAP1 / BI / 027-stammen, er opstået, forårsager epidemier af CDI 19,20. Vi har forsøgt at bestemme kliniske forløb af sygdommen, når cefoperazon-behandlede mus blev udfordret med klinisk relevant og genetisk tractable C. difficile-stamme, R20291. Denne protokol beskriver det kliniske forløb, herunder vægttab, bakteriel kolonisering, toxin cytotoksicitet, og histopatologiske ændringer i mave-tarmkanalen hos mus udfordret med C. difficile R20291 sporer. Samlet set denne musemodel viser sig at være en værdifuld eksperimentel platform for CDI tilnærme sygdom hos mennesker. Denne kendetegnet musemodel kan således anvendes til at vurdere virkningerneaf hidtil ukendte terapeutiske midler på lindring af klinisk sygdom og om genoprettelse af kolonisering modstand mod C. difficile.
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…
The authors have nothing to disclose.
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.
#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 |