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Determination of the in vitro Sporulation Frequency of Clostridium difficile
艰难梭菌孢子形成速率的体外测定   

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Abstract

The anaerobic, gastrointestinal pathogen, Clostridium difficile, persists within the environment and spreads from host-to-host via its infectious form, the spore. To effectively study spore formation, the physical differentiation of vegetative cells from spores is required to determine the proportion of spores within a population of C. difficile. This protocol describes a method to accurately enumerate both viable vegetative cells and spores separately and subsequently calculate a sporulation frequency of a mixed C. difficile population from various in vitro growth conditions (Edwards et al., 2016b).

Keywords: Clostridium difficile(艰难梭菌), Clostridium difficile infection (CDI)(艰难梭菌感染(CDI)), Anaerobe(厌氧菌), Spores(孢子), Sporulation(孢子形成), Ethanol resistance(耐乙醇性)

Background

Sporulation is a complex developmental process that results in the formation of a metabolically dormant spore. The physical properties of the C. difficile spore form provide intrinsic resistance to many environmental stresses and disinfectants, permitting its long-term survival outside of the host (reviewed in: Paredes-Sabja et al., 2014). To differentiate between the vegetative cells and spores of C. difficile, various techniques that take advantage of the physical and resistant properties of spores have been developed, including a short exposure to wet-heat or ethanol (Burns et al., 2010; Lawley et al., 2010; Edwards et al., 2014). However, these techniques may inadvertently cause long-term damage to the spores, depending on the strain of C. difficile tested, resulting in inaccurate recovery rates. Here, we describe an optimized method using a lower concentration of ethanol than previously described (40% less ethanol) to eliminate all vegetative cells within a heterogeneous C. difficile population without reducing the viability of spores. This technique provides highly reproducible and less variable results for quantifying C. difficile spore formation.

Materials and Reagents

  1. Sterile inoculating loops (Grenier Bio One, catalog number: 731170 )
  2. GeneMate 1.7 ml microcentrifuge tubes (BioExpress, catalog number: C-3260-1 )
  3. Petri dishes (94 x 16 mm) (Grenier Bio One, catalog number: 633161 )
  4. Glass test tubes with caps (18 x 150 mm) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 14-961-32 )
  5. 0.22 µm filter and syringe (for sterilization of taurocholate solutions) (CELLTREAT Scientific Products, catalog number: 229747 )
  6. 0.45 µm filter and syringe (for sterilization of D-fructose and L-cysteine solutions) (CELLTREAT Scientific Products, catalog number: 229749 )
  7. Clostridium difficile strains of interest, including the isogenic parent strain (e.g., 630Δerm or R20291) as a reference strain or positive control, test strains, and a negative control strain that is unable to sporulate, preferably in the same isogenic background as the parent (e.g., a spo0A null mutant [Heap et al., 2007; Dawson et al., 2012; Deakin et al., 2012; Fimlaid et al., 2013; Mackin et al., 2013; Edwards et al., 2014; Edwards et al., 2016a])
  8. 95% ethanol (190 proof) (Decon Labs, catalog number: 2805SG )
  9. Sterile water
  10. Taurocholate (Sigma-Aldrich, catalog number: T4009 )
  11. Brain heart infusion (BHI) (BD, catalog number: 237300 )
  12. Yeast extract (BD, catalog number: 212730 )
  13. Agar for solid medium (BD, catalog number: 214010 )
  14. Bacto peptone (BD, catalog number: 211677 )
  15. Proteose peptone (BD, catalog number: 211684 )
  16. Tris base (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP152 )
  17. Ammonium sulfate [(NH4)SO4] (Sigma-Aldrich, catalog number: A5132 )
  18. L-cysteine (Sigma-Aldrich, catalog number: C7352 )
  19. Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP358 )
  20. Potassium chloride (KCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: P217 )
  21. Sodium phosphate dibasic heptahydrate (Na2HPO4) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S373 )
  22. Potassium phosphate monobasic (KH2PO4) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP362 )
  23. D-fructose (Thermo Fisher Scientific, Fisher Scientific, catalog number: L96 )
  24. 10% (w/v) sodium taurocholate (see Recipes)
  25. Pre-reduced BHIS agar (brain heart infusion supplemented with yeast extract; see Recipes)
    Note: All media needs to be reduced before use. This is achieved by bringing plates or liquid medium into the anaerobic chamber at least 2 h for plates or overnight for liquid medium before use (for additional details, see Edwards et al., 2013).
  26. Pre-reduced BHIS agar supplemented with 0.1% taurocholate
  27. BHIS liquid medium (see Recipes)
  28. 70:30 sporulation agar (one plate per strain; see Recipes)
  29. 1x PBS (see Recipes)
  30. 20% D-fructose (see Recipes)
  31. 10% L-cysteine (see Recipes)

Equipment

  1. Anaerobic chamber (Coy Type A or Type C Chamber)
    Note: All steps are performed within the anaerobic chamber unless otherwise noted. Details on C. difficile cultivation as well as use and maintenance of an anaerobic chamber are described in (Edwards et al., 2013).
  2. Spectrophotometer (Biochrom, model: CO8000 )
  3. Autoclave

Procedure

  1. Inoculate 10 ml BHIS medium supplemented with 0.1% taurocholate and 0.2% fructose with a single C. difficile colony from a plate and incubate overnight at 37 °C. Include selective antibiotics, if necessary for plasmid maintenance.
    Note: For additional details on basic C. difficile cultivation, including propagation from frozen glycerol stocks, see Edwards et al., 2013. Taurocholate is a germinant that will promote germination of any spores present in the C. difficile colony. Fructose is an additional carbon source that prevents sporulation and reduces the accumulation of spores in the overnight culture. To ensure that the cultures are in logarithmic growth phase in the morning, an additional overnight culture of each strain may be made by back diluting the original culture 1:200-1:1,000 after inoculation into the same medium conditions.
  2. In the morning, ensure that cultures are actively growing in mid-exponential phase, e.g., the OD600 is ≤ 0.9. If cultures are past an OD600 = 0.9, and thus, entering transition or stationary phase, it may be necessary to back dilute cultures into fresh BHIS to an OD600 < 0.5 to allow the cultures to outgrow and re-enter logarithmic growth before initiating the experiment (approximately 30-90 min, depending on the original density of the overnight culture). If cultures are below OD600 < 0.9, indicating that the cells are in logarithmic growth, continue with step 3.
  3. Allow cultures to grow, or back dilute late exponential phase cultures (OD600 < 0.9) using fresh BHIS, to a final OD600 = 0.5. Apply 250 µl of culture onto the surface of fresh, pre-reduced 70:30 sporulation agar and gently spread evenly over the surface with a sterile inoculating loop. Mark the time as H0 and incubate plates for 24 h at 37 °C.
  4. Immediately perform H0 ethanol resistance sporulation assays as a control to ensure no spores are present in the exponential phase cultures. Add 0.5 ml of the back diluted (OD600 = 0.5) culture to a microcentrifuge tube containing 0.3 ml 95% ethanol and 0.2 ml dH2O. Vortex well and incubate for 15 min. Evenly spread 100 µl of each H0 control onto pre-reduced BHIS supplemented with 0.1% taurocholate agar plates and incubate at 37 °C for at least 24 h.
    Note: This control ensures that there are not a significant number of spores carried over from the overnight culture or previous passages and confirms that sporulation is not already occurring at the beginning of the sporulation assay, potentially misrepresenting the final sporulation frequencies.
  5. At H24, perform ethanol resistance sporulation assays:
    1. Enumerate vegetative cells
      1. Scrape C. difficile from the surface of the plate (approximately 1/8 of the plate, depending on the density of the cells on the plate surface) with a sterile inoculating loop and suspend cells in approximately 5 ml BHIS to an OD600 = approximately 1.0.
      2. Perform serial dilutions of suspended cells in pre-reduced BHIS liquid medium to enumerate the total number of vegetative cells. Evenly spread 100 µl onto pre-reduced BHIS agar. Typically, plating 100 µl each of 10-4 and 10-5 dilutions will yield countable colonies.
    2. Enumerate spores
      1. Aliquot 0.5 ml of culture from step 5a.i to a tube containing 0.2 ml dH2O and 0.3 ml 95% ethanol (final concentration = 28.5% ethanol). Vortex well and incubate for 15 min.
        Note: This step may be performed anaerobically or aerobically as spores are fully resistant to oxygen exposure.
      2. Serially dilute the ethanol and spore mixture from step 5b.i in 1x PBS + 0.1% taurocholate and spread 100 µl onto pre-reduced BHIS supplemented with 0.1% taurocholate plates (e.g., 100 µl of a 10-4 dilution for 630Δerm typically yields approximately 150-200 CFU). Plate as many serial dilutions as necessary to ensure countable colonies (approximately 30-400 colonies per plate).
        Note: As a control, plate 100 µl of the undiluted non-sporulating C. difficile strain after ethanol treatment (step 5b.i). This plate should yield no colony forming units (CFU), as all vegetative cells are eliminated in the presence of 28.5% ethanol. Additionally, 0.1% taurocholate must be included in the diluent and in the medium to efficiently recover all viable spores, as taurocholate serves as a germinant for C. difficile (Sorg and Sonenshein, 2008).
  6. Calculate sporulation frequency
    1. Enumerate CFU on all plates after > 24 h incubation at 37 °C. Separately calculate the total number of vegetative cells and spores, accounting for both the serial dilution and the plate dilution in the calculation.
      Note: Remember to multiply the ethanol resistant spores by a factor of 2 to account for the one-half dilution that occurs during ethanol exposure. In addition, there should be few (> 100) to no CFU on the H0 plates, and no CFU present on the spo0A mutant control plate. If more than 500 CFU are recovered at H0, this may impact the final sporulation frequency, and caution is warranted when interpreting the results. Finally, this step can be performed aerobically and with a digital colony counter to facilitate enumeration. 
    2. Calculate sporulation frequency according to equation below.


Data analysis

To calculate sporulation frequency as a percentage (%), multiply the final number by 100. Statistical analyses used will depend on the number of strains and conditions tested.

Notes

  1. In addition to determining sporulation frequency using ethanol resistance, we encourage the use of a second method, such as phase contrast microscopy (Edwards et al., 2014 and 2016b), to confirm the sporulation phenotype observed.
  2. Alternatively, this protocol may be adapted to perform sporulation assays in liquid cultures (e.g., 70:30 liquid medium, BHIS liquid medium or TY liquid medium). If these experimental conditions are desired, follow the details outlined in Edwards et al., 2014. Briefly, dilute the exponential phase culture(s) (OD600 = 0.5; from step 3) 1:10 into pre-reduced medium (the initial OD600 of the liquid cultures will be approximately 0.05). Follow growth using a spectrophotometer until the cultures reach an OD600 = 1.00, which corresponds to the beginning of transition phase for most C. difficile strains. This time point is denoted as T0. At T2-T4 (two to four hours after an OD600 = 1.00 is achieved), plate for total cells (vegetative cells and spores) present in the culture by performing serial dilutions and plating onto BHIS agar supplemented with 0.1% taurocholate. At T24, use 0.5 ml culture to perform the ethanol resistance assay as described in step 5b.i and continue the protocol as described. This method provides for the comparison of ethanol resistant spores present in the culture at T24 versus the total number of cells (vegetative cells and spores) at peak growth at T24 to calculate the sporulation frequency in liquid culture. It is important to note that much higher and more consistent sporulation frequencies are observed on plates than in liquid medium (Fimlaid et al., 2013; Edwards et al., 2016b).

Recipes

  1. 10% (w/v) sodium taurocholate
    Dissolve 1 g taurocholate in 10 ml deionized water. Sterilize with a 0.22 µm syringe filter and store at room temperature
  2. BHIS medium
    37 g/L brain heart infusion
    5 g/L yeast extract
    15 g/L agar for solid medium
    Bring to 1 L with deionized water and autoclave for 20 min at 121 °C to sterilize
    For BHIS agar supplemented with taurocholate, add 10 ml 10% taurocholate (final concentration, 0.1%) once medium cools to 60-65 °C after autoclaving
    Notes:
    1. The use of an infrared thermometer is an easy way to monitor the medium temperature.
    2. Allow medium to cool for an additional 20 min (to approximately 45-50 °C) before pouring plates. Adding the taurocholate to medium warmer than 60 °C eliminates any potential contamination from Mollicute species, which can grow on BHIS in the presence of taurocholate, a cholesterol derivative. These contaminants appear as pinpoint colonies on the surface of the agar.
  3. 70:30 sporulation medium
    63 g/L Bacto peptone
    3.5 g/L proteose peptone
    11.1 g/L brain heart infusion
    1.5 g/L yeast extract
    1.06 g/L Tris base
    0.7 g/L ammonium sulfate [(NH4)2SO4]
    15 g/L agar for solid medium
    Bring to 1 L with deionized water and autoclave for 20 min at 121 °C to sterilize. After autoclaving, add 3 ml 10% (w/v) L-cysteine (final concentration, 0.03%)
    Notes:
    1. When pouring plates, use 35 ml medium per Petri dish (94 x 16 mm) to ensure consistency between each plate.
    2. If using strains that harbor plasmids, use thiamphenicol at 2 µg/ml for maintenance. This concentration stably maintains plasmids that confer thiamphenicol resistance while not interfering with the sporulation frequency of the parent strain containing the control vector. The optimal concentration may need to be empirically determined for other antibiotics.
  4. 1x PBS
    8.01 g/L NaCl
    0.2 g/L KCl
    2.72 g/L Na2HPO4.7H2O
    0.27 g/L KH2PO4
    Bring to 1 L with deionized water and autoclave for 20 min at 121 °C or use a 0.45 µm filter to sterilize
  5. 20% D-fructose
    Dissolve 10 g D-fructose in 50 ml deionized water. Sterilize with a 0.45 µm syringe filter and store at room temperature
  6. 10% L-cysteine
    Dissolve 0.5 g L-cysteine in 5 ml deionized water. Sterilize with a 0.45 µm syringe filter. This solution precipitates and needs to be made fresh before each use

Acknowledgments

This work was supported by the U.S. National Institute of Health (NIH) grants AI116933 and AI109526 to SMM. This protocol is adapted from Edwards et al., 2016b.

References

  1. Burns, D. A., Heap, J. T. and Minton, N. P. (2010). The diverse sporulation characteristics of Clostridium difficile clinical isolates are not associated with type. Anaerobe 16(6): 618-622.
  2. Dawson, L. F., Valiente, E., Faulds-Pain, A., Donahue, E. H. and Wren, B. W. (2012). Characterisation of Clostridium difficile biofilm formation, a role for Spo0A. PLoS One 7(12): e50527.
  3. Deakin, L. J., Clare, S., Fagan, R. P., Dawson, L. F., Pickard, D. J., West, M. R., Wren, B. W., Fairweather, N. F., Dougan, G. and Lawley, T. D. (2012). The Clostridium difficile spo0A gene is a persistence and transmission factor. Infect Immun 80(8): 2704-2711.
  4. Edwards, A. N., Karim, S. T., Pascual, R. A., Jowhar, L. M., Anderson, S. E. and McBride, S. M. (2016a). Chemical and stress resistances of Clostridium difficile spores and vegetative cells. Front Microbiol 7: 1698.
  5. Edwards, A. N., Nawrocki, K. L. and McBride, S. M. (2014). Conserved oligopeptide permeases modulate sporulation initiation in Clostridium difficile. Infect Immun 82(10): 4276-4291.
  6. Edwards, A. N., Suarez, J. M. and McBride, S. M. (2013). Culturing and maintaining Clostridium difficile in an anaerobic environment. J Vis Exp(79): e50787.
  7. Edwards, A. N., Tamayo, R. and McBride, S. M. (2016b). A novel regulator controls Clostridium difficile sporulation, motility and toxin production. Mol Microbiol 100(6): 954-971.
  8. Fimlaid, K. A., Bond, J. P., Schutz, K. C., Putnam, E. E., Leung, J. M., Lawley, T. D. and Shen, A. (2013). Global analysis of the sporulation pathway of Clostridium difficile. PLoS Genet 9(8): e1003660.
  9. Heap, J. T., Pennington, O. J., Cartman, S. T., Carter, G. P. and Minton, N. P. (2007). The ClosTron: a universal gene knock-out system for the genus Clostridium. J Microbiol Methods 70(3): 452-464.
  10. Lawley, T. D., Clare, S., Deakin, L. J., Goulding, D., Yen, J. L., Raisen, C., Brantd, C., Lovell, J., Cooke, F., Clark, T. G. and Dougan, G. (2010). Use of purified Clostridium difficile spores to facilitate evaluation of health care disinfection regimens. Appl Environ Microbiol 76(20): 6895-6900.
  11. Mackin, K. E., Carter, G. P., Howarth, P., Rood, J. I. and Lyras, D. (2013). Spo0A differentially regulates toxin production in evolutionarily diverse strains of Clostridium difficile. PLoS One 8(11): e79666.
  12. Paredes-Sabja, D., Shen, A. and Sorg, J. A. (2014). Clostridium difficile spore biology: sporulation, germination, and spore structural proteins. Trends Microbiol 22(7): 406-416.
  13. Sorg, J. A. and Sonenshein, A. L. (2008). Bile salts and glycine as cogerminants for Clostridium difficile spores. J Bacteriol 190(7): 2505-2512.

简介

厌氧,胃肠道病原体,艰难梭菌在环境中持续存在,并通过其感染形式,孢子从宿主到宿主传播。为了有效地研究孢子形成,需要从孢子中进行营养细胞的物理分化以确定在C群体内孢子的比例。艰难的。该方案描述了分别精确地枚举活的营养细胞和孢子的方法,并随后计算混合的孢子形成频率。来自各种体外生长条件(Edwards等人,2016b)的难治性群体。

背景 孢子形成是一个复杂的发育过程,导致代谢休眠孢子的形成。 C的物理性质。艰难的孢子形式提供了许多环境胁迫和消毒剂的内在抵抗,允许其在宿主之外的长期生存(参见:Paredes-Sabja等人,2014年)。区分营养细胞和C孢子。已经开发了利用孢子的物理和抗性属性的各种技术,包括短时间暴露于湿热或乙醇(Burns等人,2010; Lawley&amp; et al。,2010; Edwards等人,2014)。然而,根据C的应变,这些技术可能不经意地对孢子造成长期损害。难以测试,导致恢复率不准确。在这里,我们描述了使用比以前描述的较低浓度的乙醇(40%以下的乙醇)的优化方法以消除异质C中的所有营养细胞。艰难梭菌群体,而不降低孢子的生存力。该技术为量化C提供了高度可重现性和较不可变的结果。难产孢子孢子形成。

关键字:艰难梭菌, 艰难梭菌感染(CDI), 厌氧菌, 孢子, 孢子形成, 耐乙醇性

材料和试剂

  1. 无菌接种环(Grenier Bio One,目录号:731170)
  2. GeneMate 1.7 ml微量离心管(BioExpress,目录号:C-3260-1)
  3. 培养皿(94 x 16毫米)(Grenier Bio One,目录号:633161)
  4. 玻璃试管(18 x 150 mm)(Thermo Fisher Scientific,Fisher Scientific,目录号:14-961-32)
  5. 0.22μm过滤器和注射器(用于牛磺胆酸溶液灭菌)(CELLTREAT Scientific Products,目录号:229747)
  6. 0.45μm过滤器和注射器(用于D-果糖和L-半胱氨酸溶液的灭菌)(CELLTREAT Scientific Products,目录号:229749)
  7. 感兴趣的艰难梭菌菌株,包括作为参照菌株或阳性对照的同基因亲本菌株(例如,630Δerm 或R20291),测试菌株,以及不能与母体(例如,,一个缺失突变体相同的同基因背景而不能发芽的阴性对照菌株[Heap等]/em>,2007; Dawson等人,2012; Deakin等人,2012; Fimlaid等人,2013; Mackin ,2013; Edwards等人,2014; Edwards等人,2016a])
  8. 95%乙醇(190证明)(Decon Labs,目录号:2805SG)
  9. 无菌水
  10. 牛油胆固醇(Sigma-Aldrich,目录号:T4009)
  11. 脑心输注(BHI)(BD,目录号:237300)
  12. 酵母提取物(BD,目录号:212730)
  13. 固体介质琼脂(BD,目录号:214010)
  14. Bacto蛋白胨(BD,目录号:211677)
  15. 蛋白胨(BD,目录号:211684)
  16. Tris基(Thermo Fisher Scientific,Fisher Scientific,目录号:BP152)
  17. 硫酸铵[(NH 4)SO 4](Sigma-Aldrich,目录号:A5132)
  18. L-半胱氨酸(Sigma-Aldrich,目录号:C7352)
  19. 氯化钠(NaCl)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP358)
  20. 氯化钾(KCl)(Thermo Fisher Scientific,Fisher Scientific,目录号:P217)
  21. 磷酸氢二钠七水合物(Na 2 HPO 4)(Thermo Fisher Scientific,Fisher Scientific,目录号:S373)
  22. 磷酸二氢钾(KH 2 O 3 PO 4)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP362)
  23. D-果糖(Thermo Fisher Scientific,Fisher Scientific,目录号:L96)
  24. 10%(w/v)牛磺胆酸钠(见食谱)
  25. 预先还原的BHIS琼脂(补充酵母提取物的脑心脏输注;见食谱)
    注意:使用前需要减少所有媒体。这是通过将板或液体培养基在使用前将板或液体培养基进入至少2小时至液体培养基的2小时或更长时间(参见Edwards等,2013)。
  26. 预先还原的BHIS琼脂补充0.1%牛磺胆酸盐
  27. BHIS液体培养基(见食谱)
  28. 70:30孢子琼脂(每片一片;见食谱)
  29. 1x PBS(见食谱)
  30. 20%D-果糖(见食谱)
  31. 10%L-半胱氨酸(参见食谱)

设备

  1. 厌氧室(Coy A型或C型腔)
    注意:除非另有说明,所有步骤均在厌氧室内进行。 (Edwards et al。,2013)中描述了艰难梭菌培养的细节以及厌氧室的使用和维护。
  2. 分光光度计(Biochrom,型号:CO8000)
  3. 高压灭菌器

程序

  1. 接种含有0.1%牛磺胆酸和0.2%果糖的10ml BHIS培养基。将它们从板上放置并在37℃下孵育过夜。包括选择性抗生素,必要时进行质粒维护。
    注意:有关基本的 C的其他详细信息。艰难梭菌种植,包括来自冷冻甘油储备的繁殖,参见Edwards等,2013。牛油胆酸是促进艰难梭菌菌落中存在的任何孢子的发芽的发芽剂。果糖是一种额外的碳源,可防止孢子形成并减少孢子在过夜培养中的积累。为了确保培养物在早上处于对数生长阶段,每个菌株的另外的过夜培养可以通过在接种到相同的培养基条件后将原始培养物1:200-1:1000反复稀释来进行。
  2. 早上,确保文化在中指数阶段积极生长,例如,,OD 600小于0.9。如果培养物经过OD 600 = 0.9,并因此进入转变或固定相,可能需要使稀释的培养物回到新鲜的BHIS中至OD 600, 0.5以允许培养物在开始实验之前超过并重新进入对数生长(约30-90分钟,取决于过夜培养物的原始密度)。如果培养物低于OD 600, 0.9,表示细胞处于对数生长期,继续步骤3.
  3. 允许培养物生长,或使用新鲜的BHIS将稀的后期指数期培养物(OD 600 <0.9)回升至最终的OD 600/0.5。在新鲜的预先还原的70:30孢子形琼脂的表面上加入250μl培养物,并用无菌接种环将其均匀地均匀地分散在表面上。将时间标记为H <0>,并在37℃下培养板24小时。
  4. 立即进行H <0>乙醇抗性孢子生成测定作为对照,以确保指数期培养物中不存在孢子。将0.5ml背面稀释(OD 600)= 0.5)培养物加入到含有0.3ml 95%乙醇和0.2ml dH 2 O的微量离心管中。涡旋好并孵育15分钟。将100μl每个H0.0%对照均匀地铺展到补充有0.1%牛磺胆酸琼脂平板的预还原BHIS上,并在37℃下孵育至少24小时。
    注意:该对照确保从过夜培养物或之前的传代中没有显着数量的孢子携带,并确认孢子形成测定开始时尚未发生孢子形成,可能会错误地表示最终的孢子形成频率。/em>
  5. 在H 24上进行乙醇抗性孢子生成测定:
    1. 枚举营养细胞
      1. 刮ape。使用无菌接种环从板的表面(板的大约1/8,取决于板表面上的细胞的密度),将细胞悬浮在约5ml BHIS中,以OD 600 =约1.0。
      2. 在预还原的BHIS液体培养基中进行连续稀释的悬浮细胞,以列举营养细胞的总数。将100μl均匀分散在预还原的BHIS琼脂上。通常,将10μL/sup和10×sup-5稀释液各100μl电镀得到可计数的菌落。
    2. 枚举孢子
      1. 将0.5ml来自步骤5a.i的培养物等分至含有0.2ml dH 2 O和0.3ml 95%乙醇(终浓度= 28.5%乙醇)的管中。涡旋好并孵育15分钟 注意:由于孢子完全抵抗氧气暴露,本步骤可能会厌氧或有氧地进行。
      2. 在1x PBS + 0.1%牛磺胆酸中将步骤5b.i中的乙醇和孢子混合物系统稀释,并将100μl扩散到补充有0.1%牛磺胆酸板(例如,100μl,对于630Δerm,sup> -4 稀释通常产生约150-200CFU)。根据需要将许多连续稀释物平板以确保可数的菌落(每片约30-400个菌落)。
        注意:作为对照,在乙醇处理后将100μl未稀释的未发芽的艰难梭菌菌株(步骤5b.i)平板。由于所有营养细胞在28.5%乙醇存在下被消除,所以该板不应产生菌落形成单位(CFU)。另外,0.1%牛磺胆酸盐必须包括在稀释剂和培养基中以有效地回收所有活孢子,因为牛磺胆酸作为艰难梭菌的发芽剂(Sorg和Sonenshein,2008)。
  6. 计算孢子频率
    1. 枚举所有板上的CFU>在37℃孵育24小时。分别计算营养细胞和孢子的总数,计算系列稀释度和平板稀释度。
      注意:请记住将乙醇抗性孢子乘以2,以考虑在乙醇暴露期间发生的二分之一稀释。另外,在H0平板上应该没有(> 100)至无CFU,并且spo0A突变体对照板上不存在CFU。如果超过500 CFU在H 0℃恢复,这可能会影响最终的孢子形成频率,并且在解释结果时要谨慎。最后,这个步骤可以通过有氧运动和数字殖民地计数器进行,以便于枚举。 
    2. 根据下列公式计算孢子频率

数据分析

以百分比(%)计算孢子形成频率,将最终数乘以100.所使用的统计分析将取决于测试的菌株数和条件。

笔记

  1. 除了使用乙醇抗性测定孢子频率之外,我们还鼓励使用第二种方法,如相差显微镜(Edwards等人,2014和2016b),以确认观察到的孢子形态表型。 br />
  2. 或者,该方案可以适于在液体培养物(例如,70:30液体培养基,BHIS液体培养基或TY液体培养基)中进行孢子形成测定。如果需要这些实验条件,请遵循Edwards等人,2014中概述的细节。简而言之,稀释指数期培养物(OD 600)= 0.5;来自步骤3)1:10进入预还原培养基(液体培养物的初始OD 600)将为约0.05)。使用分光光度计跟踪生长,直到培养物达到OD 600 = 1.00,其对应于大多数C的转变阶段的开始。艰难梭菌菌株。该时间点被表示为T 0> 0 。在T 2 4 <(实现OD 600> 1.00之后2-4小时),将总细胞板(营养细胞和通过进行连续稀释并电镀到补充有0.1%牛磺胆酸的BHIS琼脂上,存在于培养物中。在T 24时,使用0.5ml培养物进行如步骤5b.i中所述的乙醇抗性测定,并继续所述的方案。该方法提供了在T24亚型培养物中存在的乙醇抗性孢子与在T 24%峰值生长时的总细胞数(营养细胞和孢子)的比较,计算液体培养中的孢子形成频率。重要的是注意到在板上观察到比在液体培养基中更高和更一致的孢子频率(Fimlaid等人,2013; Edwards等人,2016b) 。

食谱

  1. 10%(w/v)牛磺胆酸钠
    将1g牛磺胆酸溶解在10ml去离子水中。用0.22μm注射器过滤器灭菌,并在室温下储存
  2. BHIS媒体
    37 g/L脑心浸液
    5克/升酵母提取物
    15 g/L琼脂固体培养基
    用去离子水冲洗至1升,121℃高压灭菌20分钟,以消毒 对于补充有牛磺胆酸盐的BHIS琼脂,一旦培养基在高压灭菌后冷却至60-65℃,加入10%10%牛磺胆酸(终浓度为0.1%), 注意:
    1. 使用加温度计是监测介质温度的简单方法
    2. 允许介质冷却另外20分钟(至约45-50℃),然后倒入板。将牛磺胆酸添加到比60℃以上的中等温度,消除了在牛磺胆酸(胆固醇衍生物)存在下可以在BHIS上生长的Mollicute物种的任何潜在污染物。这些污染物在琼脂的表面上显示为精确的菌落。
  3. 70:30孢子培养基
    63 g/L Bacto蛋白胨
    3.5 g/L蛋白胨蛋白胨
    11.1 g/L脑心脏输液 1.5 g/L酵母提取物
    1.06g/L Tris碱
    0.7g/L硫酸铵[(NH 4)2 SO 4]
    15 g/L琼脂固体培养基
    用去离子水冲洗至1升,121℃高压灭菌20分钟即可灭菌。高压灭菌后,加入3 ml 10%(w/v)L-半胱氨酸(最终浓度,0.03%) 注意:
    1. 当注射板时,每个培养皿(94 x 16 mm)使用35 ml培养基,以确保每个培养皿之间的一致性。
    2. 如果使用携带质粒的菌株,请使用2μg/ml的甲砜霉素进行维护。该浓度稳定地保持赋予甲砜霉素抗性的质粒,而不干扰含有对照载体的亲本菌株的孢子形成频率。最佳浓度可能需要经验确定其他抗生素。
  4. 1x PBS
    8.01克/升NaCl
    0.2克/升KCl
    2.72g/L Na 2 HPO 4 7H 2 O O
    0.27g/L KH 2 4
    用去离子水送至1升,121℃高压灭菌20分钟,或使用0.45微米过滤器灭菌。
  5. 20%D-果糖
    将10g D-果糖溶于50ml去离子水中。用0.45μm注射器过滤器灭菌,并在室温下储存
  6. 10%L-半胱氨酸
    将0.5g L-半胱氨酸溶于5ml去离子水中。用0.45微米注射器过滤器灭菌。这种溶液沉淀,需要在每次使用前进行清新

致谢

这项工作得到美国国家卫生研究院(NIH)向SMM授予AI116933和AI109526的支持。该协议来自于Edwards等人,2016b。

参考文献

  1. Burns,DA,Heap,JT and Minton,NP(2010)。  生物膜形成的特征,生物膜形成,Spo0A的作用。 PLoS One 7(12 ):e50527。
  2. Deakin,LJ,Clare,S.,Fagan,RP,Dawson,LF,Pickard,DJ,West,MR,Wren,BW,Fairweather,NF,Dougan,G.and Lawley,TD(2012) ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22615253"target ="_ blank">艰难梭菌spo0A 基因是一种持久性和传播性因素。感染免疫 80(8):2704-2711。
  3. Edwards,AN,Karim,ST,Pascual,RA,Jowhar,LM,Anderson,SE and McBride,SM(2016a)。  难辨梭状芽孢杆菌孢子和营养细胞的化学和抗应力。前微生物7 :1698.
  4. Edwards,AN,Nawrocki,KL和McBride,SM(2014)。一种新颖的调节剂控制艰难梭状芽孢杆菌的孢子形成,运动和毒素生成。 100微量元素(6):954-971。
  5. Fimlaid,KA,Bond,JP,Schutz,KC,Putnam,EE,Leung,JM,Lawley,TD和Shen,A.(2013)。  艰难梭菌的孢子形成途径的全球分析。 9(8):e1003660。
  6. 堆,JT,Pennington,OJ,Cartman,ST,Carter,GP和Minton,NP(2007)。 ClosTron:Clostridium属的通用基因敲除系统。 微生物方法 70(3 ):452-464。
  7. Lawley,TD,Clare,S.,Deakin,LJ,Goulding,D.,Yen,JL,Raisen,C.,Brantd,C.,Lovell,J.,Cooke,F.,Clark,TGand Dougan, (2010)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/?term=Use+of+purified+Clostridium+difficile+spores+to +促进+评估+ +健康+护理+消毒+方案"target ="_ blank">使用纯化的艰难梭菌孢子,以便于评估卫生保健消毒方案。 Appl Environ Microbiol 76(20):6895-6900。
  8. Mackin,KE,Carter,GP,Howarth,P.,Rood,JI和Lyras,D。(2013)。  艰难梭菌孢子生物学:孢子形成,萌发和孢子结构蛋白。趋势微生物 22(7):406-416。
  9. Sorg,JA和Sonenshein,AL(2008)。  胆汁 盐和甘氨酸作为难辨梭状芽胞杆菌孢子的共生菌。 J细菌 190(7):2505-2512。
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引用:Edwards, A. N. and McBride, S. M. (2017). Determination of the in vitro Sporulation Frequency of Clostridium difficile. Bio-protocol 7(3): e2125. DOI: 10.21769/BioProtoc.2125.
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