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Virulence Studies of Clostridium difficile
艰难梭菌的病毒性研究   

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Abstract

Clostridium difficile (a Gram-positive, spore-forming, strict anaerobe) can colonize antibiotic-treated hosts (McFarland, 2008). Antibiotics alter the composition of the normal, benign microbial flora which leads to loss of colonization resistance (Wilson and Perini, 1988; Antonopoulos et al., 2009). C. difficile spores germinate to actively growing bacteria which secrete toxins that damage the colonic epithelium (Voth and Ballard, 2005). The use of animal models of C. difficile disease have allowed the identification of mechanisms of colonization and virulence factors (Lyras et al., 2009; Kuehne et al., 2010; Francis et al., 2013; Aubry et al., 2012; Carter et al., 2011). This protocol describes virulence studies of C. difficile in the hamster model of C. difficile infection (Bartlett et al., 1978; Sambol et al., 2001).

Keywords: Clostridium difficile(难辨梭状芽孢杆菌), Spore(孢子), Virulence(毒力), Hamster(仓鼠), Animal model(动物模型)

Materials and Reagents

  1. Institutional Animal Care and Use Committee (IACUC)-approved animal use protocol
  2. C. difficile spores
  3. Female Syrian golden hamsters (80 g–120 g)
  4. Clindamycin Injection, USP (150 mg/ml) (Hospira, catalog number: 0409-4052 )
  5. Dulbecco’s Modified Eagle Medium (DMEM)

Equipment

  1. Animal feeding needles (gavage needles)
  2. 1 ml syringe
  3. Dedicated BSL2 animal facility
  4. Scale

Procedure

  1. Weigh each hamster.
  2. Gavage 100 μl clindamycin to each hamster (30 mg clindamycin / kg body weight).
    Note: Dilute the stock clindamycin to the appropriate concentration using DMEM.
  3. After 5 days, gavage 100 μl C. difficile spores.
    Notes:
    1. Depending on the strain of C. difficile used, the infectious dose can vary. However, in several strains, inoculation with approximately 100 spores will result in lethal disease.
    2. There are several methods to ensure reproducible infectious doses [e.g. suspending spores in phosphate buffered saline (PBS)]. Our laboratory uses Teflon-coated tubes to reduce spore adherence. Other laboratories can get reproducible infectious doses in water.
  4. Closely monitor animals for signs of infection including: wet tail, poor fur coat, lethargy, loss of 15% body weight.
    Note: Signs of disease are commonly observed in 2 – 4 days post infection. Be sure to closely monitor the animals for the above symptoms. Moribund animals can succumb to disease quickly.
  5. Humanly euthanize moribund animals, in accordance with your protocol that was approved by your Institutional Care and Animal Use Committee (IACUC). Score the time required to reach a moribund state.

Acknowledgments

We would like to thank Charlotte Allen and Ritu Shrestha for aiding in the development of this protocol. This protocol is based upon previously published C. difficile virulence studies by Sambol et al. (2001), wherein the authors analyzed the infection of hamsters by epidemiologically important C. difficile isolates. This work was supported by the American Heart Association National Scientist Development grant to JAS (No. 11SDG7160013). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  1. Antonopoulos, D. A., Huse, S. M., Morrison, H. G., Schmidt, T. M., Sogin, M. L. and Young, V. B. (2009). Reproducible community dynamics of the gastrointestinal microbiota following antibiotic perturbation. Infect Immun 77(6): 2367-2375.
  2. Aubry, A., Hussack, G., Chen, W., KuoLee, R., Twine, S. M., Fulton, K. M., Foote, S., Carrillo, C. D., Tanha, J. and Logan, S. M. (2012). Modulation of toxin production by the flagellar regulon in Clostridium difficile. Infect Immun 80(10): 3521-3532.
  3. Bartlett, J. G., Chang, T. W., Moon, N. and Onderdonk, A. B. (1978). Antibiotic-induced lethal enterocolitis in hamsters: studies with eleven agents and evidence to support the pathogenic role of toxin-producing Clostridia. Am J Vet Res 39(9): 1525-1530.
  4. Carter, G. P., Douce, G. R., Govind, R., Howarth, P. M., Mackin, K. E., Spencer, J., Buckley, A. M., Antunes, A., Kotsanas, D., Jenkin, G. A., Dupuy, B., Rood, J. I. and Lyras, D. (2011). The anti-sigma factor TcdC modulates hypervirulence in an epidemic BI/NAP1/027 clinical isolate of Clostridium difficile. PLoS Pathog 7(10): e1002317.
  5. Francis, M. B., Allen, C. A., Shrestha, R. and Sorg, J. A. (2013). Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection. PLoS Pathog 9(5): e1003356.
  6. Kuehne, S. A., Cartman, S. T., Heap, J. T., Kelly, M. L., Cockayne, A. and Minton, N. P. (2010). The role of toxin A and toxin B in Clostridium difficile infection. Nature 467(7316): 711-713.
  7. Lyras, D., O'Connor, J. R., Howarth, P. M., Sambol, S. P., Carter, G. P., Phumoonna, T., Poon, R., Adams, V., Vedantam, G., Johnson, S., Gerding, D. N. and Rood, J. I. (2009). Toxin B is essential for virulence of Clostridium difficile. Nature 458(7242): 1176-1179.
  8. McFarland, L. V. (2008). Update on the changing epidemiology of Clostridium difficile-associated disease. Nat Clin Pract Gastroenterol Hepatol 5(1): 40-48.
  9. Sambol, S. P., Tang, J. K., Merrigan, M. M., Johnson, S. and Gerding, D. N. (2001). Infection of hamsters with epidemiologically important strains of Clostridium difficile. J Infect Dis 183(12): 1760-1766.
  10. Voth, D. E. and Ballard, J. D. (2005). Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev 18(2): 247-263.
  11. Wilson, K. H. and Perini, F. (1988). Role of competition for nutrients in suppression of Clostridium difficile by the colonic microflora. Infect Immun 56(10): 2610-2614.

简介

艰难梭菌(革兰氏阳性,孢子形成,严格厌氧菌)可以定殖抗生素处理的宿主(McFarland,2008)。 抗生素改变正常的良性微生物菌群的组成,导致定殖抗性的丧失(Wilson和Perini,1988; Antonopoulos等人,2009)。 C。 艰难梭菌孢子发芽到活跃生长的细菌,其分泌破坏结肠上皮的毒素(Voth和Ballard,2005)。 使用C的动物模型。 艰难梭菌病已经允许鉴定定植和毒力因子的机制(Lyras等人,2009; Kuehne等人,2010; Francis等人, et al。,2013; Aubry et al。,2012; Carter et al。,2011)。 该方案描述了C的毒力研究。 difficile 在仓鼠模型中。 艰难梭菌感染(Bartlett等人,1978; Sambol等人,2001)。

关键字:难辨梭状芽孢杆菌, 孢子, 毒力, 仓鼠, 动物模型

材料和试剂

  1. 机构动物护理和使用委员会(IACUC)批准的动物使用协议
  2. C。 difficile 孢子
  3. 女性叙利亚金仓鼠(80g-120g)
  4. 克林霉素注射液,USP(150mg/ml)(Hospira,目录号:0409-4052)
  5. Dulbecco改良的Eagle培养基(DMEM)

设备

  1. 动物喂食针(管饲针)
  2. 1 ml注射器
  3. 专用BSL2动物设施
  4. 缩放

程序

  1. 称重每只仓鼠。
  2. 向每只仓鼠灌注100μl克林霉素(30mg克林霉素/kg体重) 注意:使用DMEM稀释股票克林霉素至适当浓度。
  3. 5天后,管饲100μl。 艰难梭菌孢子。
    注意:
    1. 根据所用的艰难梭菌的菌株,感染剂量可以变化。 然而,在几个菌株中,接种约100个孢子将导致致死性疾病。
    2. 有几种方法可确保可重复的感染剂量[例如悬浮孢子在磷酸盐缓冲盐水(PBS)中]。我们的实验室使用特氟龙涂层管减少孢子粘附。其他实验室可在水中获得可重现的感染剂量。
  4. 仔细监控动物的感染征象,包括:湿尾,皮毛大衣不舒服,嗜睡,体重减轻15%。
    注意:疾病的体征通常在感染后2-4天观察到。一定要密切监测动物的上述症状。 Moribund动物可以迅速屈服于疾病。
  5. 根据您的机构护理和动物使用委员会(IACUC)批准的协议,人类安乐死濒死的动物。计算达到垂死状态所需的时间。

致谢

我们要感谢Charlotte Allen和Ritu Shrestha帮助开发这个协议。该协议基于以前公布的C。艰难梭菌毒力研究(Sambol等人,(2001), 其中作者通过流行病学上重要的C分析了仓鼠的感染。艰难梭菌分离株。这项工作是由美国心脏协会国家科学家发展赠款给JAS(No. 11SDG7160013)支持。资助者在研究设计,数据收集和分析,决定发布或准备手稿方面没有任何作用。

参考文献

  1. Antonopoulos,D.A.,Huse,S.M.,Morrison,H.G.,Schmidt,T.M.,Sogin,M.L.and Young,V.B。(2009)。 抗生素扰动后胃肠道微生物群落的可重现的社区动力学 /em> 77(6):2367-2375。
  2. Aubry,A.,Hussack,G.,Chen,W.,KuoLee,R.,Twine,S.M.,Fulton,K.M.,foote,S.,Carrillo,C.D.,Tanha,J.and Logan, 由艰难梭菌中的鞭毛调节子调节毒素产生。 a> Infect Immun 80(10):3521-3532。
  3. Bartlett,J.G.,Chang,T.W.,Moon,N。和Onderdonk,A.B。(1978)。 仓鼠的抗生素致死性小肠结肠炎:使用十一种药剂和 支持毒素产生梭菌的病原作用的证据。 Am J Vet Res 39(9):1525-1530。
  4. Carter,GP,Douce,GR,Govind,R.,Howarth,PM,Mackin,KE,Spencer,J.,Buckley,AM,Antunes,A.,Kotsanas,D.,Jenkin,GA,Dupuy, ,JI和Lyras,D。(2011)。 抗西格玛因子TcdC调节流行性BI/NAP1/027临床分离株中的高毒性>艰难梭菌。 PLoS Pathog 7(10):e1002317。
  5. Francis,M.B.,Allen,C.A.,Shrestha,R。和Sorg,J.A。(2013)。 由艰难梭菌发芽受体(CspC)识别胆汁酸是重要的用于建立感染。 PLoS Pathog 9(5):e1003356。
  6. Kuehne,S.A.,Cartman,S.T.,Heap,J.T.,Kelly,M.L.,Cockayne,A.and Minton,N.P。(2010)。 艰难梭菌感染中毒素A和毒素B的作用。/a> 自然 467(7316):711-713
  7. Lyras,D.,O'Connor,JR,Howarth,PM,Sambol,SP,Carter,GP,Phumoonna,T.,Poon,R.,Adams,V.,Vedantam, DN和Rood,JI(2009)。 毒素B对难辨梭状芽孢杆菌的毒力至关重要。 Nature 458(7242):1176-1179。
  8. McFarland,L.V。(2008)。 关于艰难梭菌相关疾病的流行病学变化的更新。 Nat Clin Pract Gastroenterol Hepatol 5(1):40-48。
  9. Sambol,S.P.,Tang,J.K.,Merrigan,M.M.,Johnson,S.and Gerding,D.N。(2001)。 感染具有流行病学重要的艰难梭菌菌株的仓鼠。 J Infect Dis 183(12):1760-1766。
  10. Voth,D.E。和Ballard,J.D。(2005)。 艰难梭菌毒素:作用机制和在疾病中的作用。 a> Clin Microbiol Rev 18(2):247-263。
  11. Wilson,K.H。和Perini,F。(1988)。 结肠微生物群对竞争抑制艰难梭菌的营养素的作用。 Infect Immun 56(10):2610-2614。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Francis, M. B. and Sorg, J. A. (2013). Virulence Studies of Clostridium difficile. Bio-protocol 3(24): e1002. DOI: 10.21769/BioProtoc.1002.
  2. Francis, M. B., Allen, C. A., Shrestha, R. and Sorg, J. A. (2013). Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection. PLoS Pathog 9(5): e1003356.
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