该协议概述了使用临床相关和转听话应变,R20291 艰难梭菌感染(CDI)的头孢哌酮小鼠模型。临床疾病的监测, 艰难梭菌细菌枚举,毒素的细胞毒作用,并在小鼠模型中在整个CDI病理学改变强调在协议中详述。
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.
艰难梭菌是一种厌氧,革兰氏阳性,形成孢子的杆菌引起危及生命的腹泻1。 艰难梭菌感染(CDI)与日益增多的人类发病率和死亡率,并导致每年1-4医疗费用相关的超过480十亿$。在2013年,美国疾病控制和预防归类难辨作为一项紧迫抗生素耐药性的风险,这表明它对公众健康的1紧迫的威胁。目前,万古霉素抗生素治疗和甲硝唑被认为是照顾的CDI 5标准。不幸的是,CDI复发成功治疗后用抗生素为高,在20发生-的患者2,5-7 30%。因此,新的治疗剂的发现对这种肠道病原体是必要的。为了评估对难辨梭状芽孢杆菌 ,动物模型近似交流人类疾病治疗需要linically相关的应变。
最初,柯赫氏法则是在1977年用克林霉素治疗的叙利亚仓鼠模型建立8 艰难梭菌 。这种模式是今天仍然利用探讨发病9,10的难辨梭状芽孢杆菌毒素的作用。然而,CDI在仓鼠模型导致高死亡率和不近似慢性阴险疾病表现可与CDI 10,11人看到。根据在研究鼠平台的辅助和试剂可用性,CDI的小鼠模型是相关的。
在2008年,CDI的健壮小鼠模型,通过用在饮用水3天之后克林霉素12腹膜内注射抗生素混合物(卡那霉素,庆大霉素,粘菌素,甲硝唑,万古霉素)治疗的小鼠建立的。这种小鼠呈现易受CDI和严重的结肠炎。依靠ING上施用接种的剂量,可以使用该模型被观察到的范围内的临床症状和杀伤力。因为这个时候,各个抗生素疗法已调查了改变的鼠肠道菌群,降低到艰难梭菌可以定植在胃肠道的点定植抗力(在最佳等人审查。和罗礼&Young的)13,14。
最近,一种广谱头孢菌素,头孢哌酮,饮用水中的给定的5天或10天重复地呈现小鼠易感CDI 15。由于第三代头孢菌素的管理都与人类的CDI的风险增加,使用头孢哌酮模型更准确地反映自然发生的疾病16。头孢哌酮治疗小鼠容易艰难梭菌受到挑战既艰难梭菌芽孢和范围在临床多种菌株的营养细胞相关性和毒力17。尽管出现了一些利用难辨营养细胞的感染形式的原始研究, 艰难梭菌芽孢被认为是传动装置18的主要方式。
在过去十年中, 艰难梭菌 R20291,一个NAP1 / BI / 027株,已经出现,造成CDI 19,20流行。我们试图确定疾病的临床过程时头孢哌酮处理的小鼠用临床相关和基因-易于处理艰难梭菌菌株,R20291挑战。该协议细节的临床过程,包括体重减轻,细菌定植,毒素的细胞毒性,并与艰难梭菌 R20291孢子攻击的小鼠的胃肠道组织病理学变化。总体而言,这种小鼠模型被证明是CDI近似人类疾病的宝贵的实验平台。此其特征小鼠模型因此可用于评估的影响对临床疾病的改善和关于对艰难梭菌定植抗力的恢复新颖治疗剂。
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 |