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Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine
用于小鼠肠道梭菌增菌培养的孢子制备方案   

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Abstract

In recent years, many spore-forming commensal Clostridia found in the gut have been discovered to promote host physiology, immune development, and protection against infections. We provide a detailed protocol for rapid enrichment of spore-forming bacteria from murine intestine. Briefly, contents from the intestinal cecum are collected aerobically, diluted and finally treated with chloroform to enrich for Clostridia spores.

Keywords: Spores(孢子), Clostridia(梭菌), Bacteria(细菌), Mammalian(哺乳动物), Murine(鼠), Intestine(肠道), Colonization resistance(定植抗力)

Background

Chloroform kills vegetative bacterial cells but not spores and thus is a useful treatment for enriching Clostridia, the dominant spore-forming group in the mammalian intestine. Experimental procedures for chloroform treatment of mouse feces exist (Momose et al., 2009; Yano et al., 2015). However, they utilize specialized equipment including an anaerobic chamber. We realized that several brief exposures to oxygen occur during experimental manipulation of intestinal contents in preparation for and after chloroform treatment. Therefore, we reasoned the sufficient recover of murine spore-forming bacteria could be obtained without the use of an anaerobic chamber. Since spore-forming Clostridia are a dominant species in the mammalian intestine, this protocol could potentially be used for isolation of spores from the intestines of other mammalian organisms, including larger rodents, primates, and humans.

Materials and Reagents

  1. Sterile 1.5 ml microcentrifuge tubes (Eppendorf, catalog number: 022363204 )
  2. 15 ml tubes
  3. Female (male mice may be used if necessary) C57BL/6 mice aged 8-12 weeks (THE JACKSON LABORATORIES)
  4. Compressed CO2 gas in cylinder (AirGas)
  5. Chloroform (Sigma-Aldrich, catalog number: 288306 )
  6. Sterile PBS pH 7.4 (Reference 2)

Equipment

  1. Sterile necropsy instruments (operating scissors, tweezers and forceps) to avoid contamination
  2. Shaker with 200 rpm capacity and 37 °C setting (or inside 37 °C room)

Procedure

  1. Without pre-charging the chamber, place the animal(s) in the chamber and introduce 100% carbon dioxide (whenever possible euthanize animals in their home cage). Open the CO2 tank or valve regulator to initiate flow of gas. A fill rate of about 10% to 30% of the chamber volume per minute with carbon dioxide, added to the existing air in the chamber should be appropriate to achieve a balanced gas mixture to fulfill the objective of rapid unconsciousness with minimal distress to the animals. Wait approximately 3-5 min for animal to stop moving or breathing. Maintain CO2 flow for a minimum of 1 min after respiration ceases. Death was confirmed by cervical dislocation.
  2. Open the abdominal cavity using operating scissors to remove the cecum. Using operating scissors, keep the cecum intact by carefully excising to not lose any contents.
  3. Open the cecum with operating scissors and add the cecal contents to a 1.5 ml microcentrifuge tube on ice.
  4. Take cecal contents and dilute 1:10 in PBS (w/v). Add chloroform in a final concentration of 3% (v/v).
  5. Incubate the cecal contents/chloroform mixture by shaking at 200 rpm at 37 °C for 30 min.
  6. Allow the chloroform to settle to the bottom of the tube at room temperature (approximately 20 min).
  7. Remove top aqueous layer to recover the spores and add to a sterile 1.5 ml tube. Be careful not to take any chloroform that has settled to the bottom of the tube.

Data analysis

Real-Time PCR using Clostridia specific primers were used for subsequent analysis of log CFU of Clostridia in feces of mice that received spores isolated by this protocol (Rivera-Chávez et al., 2016). Fold changes of ratios (mRNA levels) were transformed logarithmically prior to statistical analysis. An unpaired Student’s t-test was used to determine whether differences in fold changes between groups were statistically significant (P < 0.05).

Notes

  1. Expect 100 to 200 mg of cecal contents per mouse.
  2. It is very important to collect the cecal contents as quickly as possible once mouse has been euthanized to ensure maximum recovery of spores.
  3. Use sterile technique to not contaminate cecal contents during transfer into a sterile 1.5 ml tube.
  4. If pooled cecal contents from multiple mice will be prepared, then 15 ml tubes could be used to accommodate greater volumes.
  5. Isolated spores should be used fresh (same day of isolation) to ensure maximum viability of spores. We do not recommend storing spores.

Acknowledgments

This protocol was adapted from Momose et al., 2009. This work was supported by Public Health Service Grants AI096528 (A.J.B.), AI112949 (A.J.B.), AI103248 (S.E.W.), AI112241 (C.A.L.), OD010931 (E.M.V.), and AI060555 (E.M.V. and F.R.-C.).

References

  1. Momose, Y., Maruyama, A., Iwasaki, T., Miyamoto, Y. and Itoh, K. (2009). 16S rRNA gene sequence-based analysis of clostridia related to conversion of germfree mice to the normal state. J Appl Microbiol 107(6): 2088-2097.
  2. Phosphate-buffered saline (PBS) (2006). Cold Spring Harb Protoc.
  3. Rivera-Chávez, F., Zhang, L. F., Faber, F., Lopez, C. A., Byndloss, M. X., Olsan, E., Xu, G., Velazquez, E. M., Lebrilla, C., Winter, S. E. and Bäumler, A. J. (2016). Depletion of butyrate-producing Clostridia from the gut microbiota drives an aerobic luminal expansion of Salmonella. Cell Host Microbe 19: 443-454.
  4. Yano, J. M., Yu, K., Donaldson, G. P., Shastri, G. G., Ann, P., Ma, L., Nagler, C. R., Ismagilov, R. F., Mazmanian, S. K. and Hsiao, E.Y. (2015). Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell 161(2): 264-276.

简介

近年来,发现肠道中发现的许多孢子形成共生梭菌可以促进宿主的生理,免疫发育和防止感染。我们提供了一个详细的方案,用于从鼠肠中快速富集孢子形成细菌。简单地说,盲肠内容物有氧地进行收集,稀释,最后用氯仿处理以丰富梭菌孢子。

背景 氯仿杀死营养细菌而不是孢子,因此是丰富梭菌(Clostridia)的有效方法,梭菌是哺乳动物肠道中主要的孢子形成组。存在用于小鼠粪便的氯仿处理的实验程序(Momose等人,2009; Yano等人,2015)。然而,他们利用专门的设备,包括厌氧室。我们意识到在氯仿处理和氯仿处理过程中,肠内容物的实验操作过程中发生几次短暂的氧气暴露。因此,我们认为可以在不使用厌氧室的情况下获得足够的鼠孢子形成细菌的恢复。由于孢子形成梭菌是哺乳动物肠道中的优势物种,因此该方案可能潜在地用于从其他哺乳动物生物体(包括较大的啮齿动物,灵长类动物和人类)的肠道中分离出孢子。

关键字:孢子, 梭菌, 细菌, 哺乳动物, 鼠, 肠道, 定植抗力

材料和试剂

  1. 无菌1.5ml微量离心管(Eppendorf,目录号:022363204)
  2. 15 ml管子
  3. 女性(如果需要,可以使用雄性小鼠)8-12周龄的C57BL / 6小鼠(THE JACKSON LABORATORIES)
  4. 气瓶(AirGas)中压缩的CO 2气体
  5. 氯仿(Sigma-Aldrich,目录号:288306)
  6. 无菌PBS pH 7.4(参考文献2)

设备

  1. 无菌尸检仪器(操作剪刀,镊子和镊子)避免污染
  2. 振荡器具有200转/分钟的容量和37°C的设置(或37°C的室内)

程序

  1. 不预充气室,将动物放入室内并引入100%的二氧化碳(只要可能,将家畜笼中的动物安乐死)。打开CO 2子罐或阀调节器以启动气体流。添加到室内现有空气中的二氧化碳每分钟约10%至30%的填充率应适合于实现平衡的气体混合物,以达到快速无意识的目的,同时对动物的痛苦最小。等待约3-5分钟,动物停止移动或呼吸。呼吸停止后至少1分钟保持CO 2次流。死亡被宫颈脱位证实。
  2. 使用操作剪刀打开腹腔以去除盲肠。使用操作剪刀,通过仔细切除以保持盲肠完好无损,不丢失任何内容。
  3. 用操作剪刀打开盲肠,并将盲瓶内容物加入到冰上的1.5ml微量离心管中。
  4. 取盲肠内容物,并稀释1:10在PBS(w / v)。加入终浓度为3%(v / v)的氯仿。
  5. 通过在37℃下以200rpm摇动30分钟来孵育盲肠内容物/氯仿混合物
  6. 在室温(约20分钟)下使氯仿沉淀到管的底部。
  7. 去除顶层水层以回收孢子并加入无菌的1.5ml管中。小心不要取任何已经沉降到管底部的氯仿。

数据分析

使用Clostridia特异性引物的实时PCR用于随后分析通过该方案分离的接种孢子的小鼠粪便中梭菌的对数CFU(Rivera-Chávez等人,2016)。在统计分析之前,将对比的折叠变化(mRNA水平)进行对数转换。使用不配对的Student's 测试来确定组之间的折叠变化的差异是否具有统计学意义(P <0.05)。

笔记

  1. 每只小鼠每天要排出100〜200毫克的盲肠内容物
  2. 一旦鼠标已被安乐死以确保孢子的最大回收率,收集盲肠内容物是非常重要的。
  3. 使用无菌技术在转移到无菌的1.5 ml管中时不会污染盲肠内容物
  4. 如果将准备来自多个小鼠的汇集的盲肠内容物,则可以使用15ml管来容纳更大的体积。
  5. 隔离的孢子应该是新鲜的(隔离当天),以确保孢子的最大生存力。我们不建议储存孢子。

致谢

该工作由公共卫生服务授权AI096528(AJB),AI112949(AJB),AI103248(SEW),AI112241(CAL),OD010931(CALI)等提供支持。 EMV)和AI060555(EMV和FR-C)。

参考

  1. Momose,Y.,Maruyama,A.,Iwasaki,T.,Miyamoto,Y.and Itoh,K。(2009)。&lt; a class ="ke-insertfile"href ="https://www.ncbi。 nlm.nih.gov/pubmed/19614852"target ="_ blank"> 16S rRNA基因序列分析与无菌小鼠转化为正常状态有关的梭菌 .J Appl Microbiol 107(6):2088-2097。
  2. 磷酸盐缓冲盐水(PBS)(2006)。冷泉Harb Protoc 。
  3. Rivera-Chávez,F.,Zhang,LF,Faber,F.,Lopez,CA,Byndloss,MX,Olsan,E.,Xu,G.,Velazquez,EM,Lebrilla,C.,Winter,SE andBäumler,AJ (2016)。从肠道微生物群中消耗产生丁酸梭菌的梭菌驱动沙门氏菌的有氧管腔扩张。细胞宿主微生物 19: 443-454。
  4. Yano,J.M.,Yu,K.,Donaldson,G.P.Shastri,G.G.,Ann,P.,Ma,L.,Nagler,C.R.,Ismagilov,R.F.,Mazmanian,S.K。和Hsiao,E.Y。 (2015)。来自肠道微生物群的土着细菌调节宿主血清素生物合成。细胞 161(2):264-276。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Velazquez, E. M., Rivera-Chávez, F. and Bäumler, A. J. (2017). Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine. Bio-protocol 7(10): e2296. DOI: 10.21769/BioProtoc.2296.
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