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Adherent-invasive Escherichia coli Biofilm Formation Assays
附着侵袭大肠杆菌生物膜形成试验   

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Abstract

Patients with Crohn’s disease are abnormally colonized by adherent-invasive Escherichia coli (AIEC) bacteria (Chassaing and Darfeuille-Michaud, 2011). These bacteria are able to adhere to and invade intestinal epithelial cells (IEC), to replicate within macrophages, and were recently described to be able to form biofilms (Martinez-Medina et al., 2009; Chassaing and Darfeuille-Michaud, 2013). The reference strain of adherent-invasive E. coli is the strain LF82, associated with ileal Crohn’s disease (Darfeuille-Michaud et al., 1998).
This protocol described basic steps of a biofilm formation assay on I) non-cell-treated polystyrene microtiter plates and on II) paraformaldehyde-fixed I-407 IEC monolayers.

Keywords: Inflammatory Bowel Disease(炎症性肠病), Adherent-Invasive Escherichia coli(贴壁侵袭性大肠杆菌), Biofilm(生物膜)

Materials and Reagents

  1. Adherent-invasive E. coli reference strain LF82 (Darfeuille-Michaud et al., 1998)
  2. Intestine-407 (I-407) cells (ATCC, catalog number: CCL-6 )
  3. Luria Broth (BD DifcoTM, catalog number: 244620 )
  4. Glucose (Sigma-Aldrich, catalog number: G8270 )
  5. M63 medium (United States Biological, catalog number: M1015 )
  6. MgSO4 (Sigma-Aldrich, catalog number: 208094 )
  7. Phosphate-Buffered Saline (PBS) ( Mediatech, Cellgro®, catalog number: 21-040 )
  8. Crystal Violet (Sigma-Aldrich, catalog number: C6158 )
  9. 200 proof absolute Ethanol (Sigma-Aldrich)
  10. Fetal bovine serum (FBS) (Mediatech, Cellgro®, catalog number: 35-010-CV )
  11. Nonessential amino acids (Mediatech, Cellgro®, catalog number: 25-025-CI )
  12. L-glutamine (Mediatech, Cellgro®, catalog number: 25-005 )
  13. Penicillin/Streptomycin/Amphotericin B solution (Mediatech, Cellgro®, catalog number: 30-004-CI )
  14. Vitamin mix (Mediatech, Cellgro®, catalog number: 25-020-CI )
  15. Formaldehyde solution (Sigma-Aldrich, catalog number: F8775 )
  16. Phalloidin-tetramethyl rhodamine isocyanate (Sigma-Aldrich, catalog number: P1951 )
  17. Paraformaldehyde
  18. Hoechst 33258 (Sigma-Aldrich, catalog number: B1155 )
  19. Vectashield (Vector Labs, catalog number: H-1000 )
  20. Minimum Essential Medium (MEM) (Mediatech, Cellgro®, catalog number: 10-022-CV ) (see Recipes)

Equipment

  1. Coverslips (Electron Microscopy Sciences)
  2. Sterile tubes
  3. 24-well tissue culture plates
  4. Non-cell-treated polystyrene 96-well microtiter plates (Falcon®, catalog number: 62406-117 )
  5. 24-well polystyrene plate, tissue-culture treated (Falcon®, catalog number: 62406-159 )
  6. Microplate shaker (LABREPCO, model: BT1500 )
  7. Microplate spectrophotometer
  8. Confocal microscope
  9. 30 °C incubator
  10. 37 °C/5% CO2 incubator

Software

  1. Computer program COMSTAT1

Procedure

  1. Biofilm formation assay on non-cell-treated polystyrene microtiter plates
    1. Bacterial strains were grown overnight in sterile tubes containing 2 ml of Luria-Bertani broth with 5 g.L-1 glucose at 35.5 °C without agitation (Martinez-Medina et al., 2009; Chassaing and Darfeuille-Michaud, 2013).
    2. The following day, bacterial suspension was diluted 1/100 in M63 minimal medium supplemented with 1 mM of MgSO4 and 8 g.L-1 glucose.
    3. Aliquots of 130 μl were then placed in wells of non-cell-treated polystyrene 96-well microtiter plates and incubated 24 h at 30 °C without shaking. One well designed as blank received only M63 minimal medium supplemented with 1 mM of MgSO4 and 8 g.L-1 glucose without bacteria.
    4. After incubation, microplates were agitated for 5 min using a microplate shaker, and bacterial growths were then estimated by OD620nm reading using a microplate spectrophotometer.
    5. The liquid was remove by decantation, and the wells were washed once using 200 μl sterile PBS without agitation.
    6. In order to dry them, plates were left open for at least 20 min at room temperature.
    7. Adherent bacteria forming biofilm were stained with 130 μl of 1% crystal violet solubilized in ethanol for 5 min at room temperature without agitation.
    8. Wells were then washed five times using 200 μl sterile PBS without agitation.
    9. In order to dry them, plates were left open for at least 1 h at room temperature.
    10. 130 μl of absolute ethanol was added, and plates were incubated 5 min at room temperature without agitation.
    11. High-speed agitation was performed using a microplate shaker for 10 min (1000 rpm).
    12.  Biofilm formation was estimated by OD570nm reading using a microplate spectrophotometer.
      13. Specific biofilm formation index could be determined by using the ration OD570nm/OD620nm that allows standardization of biofilm formation according to bacterial growth in M63 minimal medium. According to previous publication, specific biofilm formation index of wild type AIEC strain LF82 should be between 2 and 4 (Martinez-Medina et al., 2009; Chassaing and Darfeuille-Michaud, 2013).

  2. Biofilm formation assay on paraformaldehyde-fixed I-407 IEC monolayers
    Intestine-407 (I-407) cells were derived from human intestinal embryonic jejunum and ileum tissues. They were maintained in an atmosphere containing 5% CO2 at 37 °C in Minimum Essential Medium supplemented with 10% (vol/vol) fetal bovine serum; 1% of nonessential amino acids; 1% of L-glutamine; 1% of Penicillin/Streptomycin/Amphotericin B solution and 1% of vitamin mix.
    1. Monolayers were seeded on coverslip in 24-well tissue culture plates with 4 x 105 cells per well and incubated for 20 h at 37 °C with 5% CO2.
    2. Monolayers were then washed once with 1ml PBS, fixed for 15 min in 4% formaldehyde (stock solution diluted in sterile PBS) at room temperature without agitation, and then washed 4 times with PBS.
    3. Bacterial strains expressing green fluorescent protein (GFP) (Valdivia et al., 1996) were prepared as previously described above, from step A1 to A2, in M63 minimal medium supplemented with 1 mM of MgSO4 and 8 g.L-1 glucose.
    4. 1 ml of this bacterial suspension was applied on the surface of fixed I-407 cell monolayers, and incubated overnight at 30 °C without shaking.
    5. The liquid was removed by decantation, and the wells were washed 3 times using 1 ml of sterile PBS without incubation.
    6. I-407/biofilm complex was then fixed for 15 min using 4% formaldehyde.
    7. Phalloidin-tetramethyl rhodamine isocyanate was used to visualize actin and Hoechst 33258 was used to visualize nuclei. For this purpose, Phalloidin-tetramethyl rhodamine isocyanate was diluted in sterile PBS at a final concentration of 1μg/ml and Hoechst 33258 was added at a final concentration of 1μg/ml. This solution was added to the wells and incubated for 20 min at room temperature. After incubation, the wells were washed 5 times using 1 ml of sterile PBS without incubation.
    8. Coverslips containing the fixed and stained I-407/biofilm complex were then removed from the 24-well plate and mounted on slide using Vectashield.
    9. The slides were examined with a confocal microscope. An example of biofilm formation on paraformaldehyde-fixed I-407 IEC monolayers is presented Figure 1.
    10. Images from biofilm formation assay at the surface of intestinal epithelial cells I-407 monolayers could be analyzed for thickness and/or roughness with the computer program COMSTAT1 (Haydorn et al., 2000).


      Figure 1. Confocal analysis of biofilm formation of a poor biofilm producer (Non pathogenic Escherichia coli K12 strain C600 (A) and a strong biofilm producer (Adherent-Invasive Escherichia coli strain LF82 (B) at the surface of a PFA-fixed monolayer of I-407 intestinal epithelial cells. Bacteria express GFP (green), actin is stained in red using phalloidin-TRITC, and DNA is stained in blue using Hoechst. Bars, 50 m.

Notes

  1. Due to high variation observed with both of these biofilm formation assays, they need to be performed at least in triplicate, including controls.

Recipes

  1. Minimum Essential Medium supplemented with
    10% (vol/vol) fetal bovine serum
    1% of nonessential amino acids
    1% of L-glutamine
    1% of Penicillin/Streptomycin/Amphotericin B solution
    1% of vitamin mix

Acknowledgments

This study was supported by the Ministère de la Recherche et de la Technologie, the Institut National de la Santé et de la Recherche Médicale and the Université d’Auvergne (UMR Inserm 1071), the Institut National de la Recherche Agronomique (USC INRA 2018) and by grants from the Association F. Aupetit (AFA). We thank the CICS platform for confocal microscopy. This protocol is adapted from previously published papers (Danese et al., 2000; Naves et al., 2008; Martinez-Medina et al., 2009; Chassaing and Darfeuille-Michaud, 2013). This work is dedicated to Arlette Darfeuille-Michaud.

References

  1. Chassaing, B. and Darfeuille-Michaud, A. (2011). The commensal microbiota and enteropathogens in the pathogenesis of inflammatory bowel diseases. Gastroenterology 140(6): 1720-28.
  2. Chassaing, B. and Darfeuille-Michaud, A. (2013). The sigmaE pathway is involved in biofilm formation by Crohn's disease-associated adherent-invasive Escherichia coli. J Bacteriol 195(1): 76-84.
  3. Danese, P. N., Pratt, L. A., Dove, S. L. and Kolter, R. (2000). The outer membrane protein, antigen 43, mediates cell-to-cell interactions within Escherichia coli biofilms. Mol Microbiol 37(2): 424-432.
  4. Darfeuille-Michaud, A., Neut, C., Barnich, N., Lederman, E., Di Martino, P., Desreumaux, P., Gambiez, L., Joly, B., Cortot, A. and Colombel, J. F. (1998). Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease. Gastroenterology 115(6): 1405-1413.
  5. Martinez-Medina, M., Naves, P., Blanco, J., Aldeguer, X., Blanco, J. E., Blanco, M., Ponte, C., Soriano, F., Darfeuille-Michaud, A. and Garcia-Gil, L. J. (2009). Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC). BMC Microbiol 9: 202.
  6. Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K. and Molin, S. (2000). Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146 ( Pt 10): 2395-2407. 
  7. Naves, P., del Prado, G., Huelves, L., Gracia, M., Ruiz, V., Blanco, J., Dahbi, G., Blanco, M., Ponte Mdel, C. and Soriano, F. (2008). Correlation between virulence factors and in vitro biofilm formation by Escherichia coli strains. Microb Pathog 45(2): 86-91.
  8. Valdivia, R. H., Hromockyj, A. E., Monack, D., Ramakrishnan, L. and Falkow, S. (1996). Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions. Gene 173(1 Spec No): 47-52.

简介

克罗恩病患者被粘附侵入性大肠杆菌(AIEC)细菌异常定植(Chassaing和Darfeuille-Michaud,2011)。 这些细菌能够粘附和侵入肠上皮细胞(IEC),在巨噬细胞内复制,并且最近被描述为能够形成生物膜(Martinez-Medina等人,2009; Chassaing和 Darfeuille-Michaud,2013)。 粘附侵入性E的参考应变。 大肠杆菌是与回肠克罗恩病相关的菌株LF82(Darfeuille-Michaud等人,1998)。
该方案描述了生物膜形成测定的基本步骤:I) 非细胞处理的聚苯乙烯微量滴定板和II)多聚甲醛固定的I-407 IEC单层。

关键字:炎症性肠病, 贴壁侵袭性大肠杆菌, 生物膜

材料和试剂

  1. 粘附侵入性 大肠杆菌参考菌株LF82(Darfeuille-Michaud等人,1998)
  2. 肠407(I-407)细胞(ATCC,目录号:CCL-6)
  3. Luria Broth(BD Difco TM ,目录号:244620)
  4. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  5. M63培养基(United States Biological,目录号:M1015)
  6. MgSO 4(Sigma-Aldrich,目录号:208094)
  7. 磷酸盐缓冲盐水(PBS)(Mediatech,Cellgro ,目录号:21-040)
  8. 结晶紫(Sigma-Aldrich,目录号:C6158)
  9. 200 proof绝对乙醇(Sigma-Aldrich)
  10. 胎牛血清(FBS)(Mediatech,Cellgro ,目录号:35-010-CV)
  11. 非必需氨基酸(Mediatech,Cellgro ,目录号:25-025-CI)
  12. L-谷氨酰胺(Mediatech,Cellgro ,目录号:25-005)
  13. 青霉素/链霉素/两性霉素B溶液(Mediatech,Cellgro ,目录号:30-004-CI)
  14. 维生素混合物(Mediatech,Cellgro ,目录号:25-020-CI)
  15. 甲醛溶液(Sigma-Aldrich,目录号:F8775)
  16. 鬼笔环胺 - 四甲基若丹明异氰酸酯(Sigma-Aldrich,目录号:P1951)
  17. 多聚甲醛
  18. Hoechst 33258(Sigma-Aldrich,目录号:B1155)
  19. Vectashield(Vector Labs,目录号:H-1000)
  20. 最低必需培养基(MEM)(Mediatech,Cellgro ,目录号:10-022-CV)(参见配方)

设备

  1. 盖玻片(电子显微镜科学)
  2. 无菌管
  3. 24孔组织培养板
  4. 非细胞处理的聚苯乙烯96孔微量滴定板(Falcon ,目录号:62406-117)
  5. 24孔聚苯乙烯板,组织培养处理(Falcon ,目录号:62406-159)
  6. 微量培养板振荡器(LABREPCO,型号:BT1500)
  7. 微孔板分光光度计
  8. 共焦显微镜
  9. 30℃培养箱
  10. 37℃/5%CO 2培养箱

软件

  1. 计算机程序COMSTAT1

程序

  1. 在非细胞处理的聚苯乙烯微量滴定板上的生物膜形成测定
    1. 细菌菌株在无搅拌下在含有2ml含有5gL -1 -1葡萄糖的Luria-Bertani肉汤的无菌管中在35.5℃下生长过夜(Martinez-Medina等人, 2009; Chassaing和Darfeuille-Michaud,2013)。
    2. 第二天,将细菌悬浮液在补充有1mM MgSO 4和8gL -1葡萄糖的M63基本培养基中稀释1/100。
    3. 然后将130μl的等分试样置于非细胞处理的聚苯乙烯96孔微量滴定板的孔中,并在30℃下不摇动孵育24小时。一个设计为空白的设计仅接受补充有1mM MgSO 4和8gL -1葡萄糖而不含细菌的M63基本培养基。
    4. 孵育后,使用微孔板振荡器将微孔板搅拌5分钟,然后使用微孔板分光光度计通过OD 620nm读数估计细菌生长。
    5. 通过倾析除去液体,用200μl无菌PBS洗涤孔一次,无搅拌
    6. 为了干燥它们,将板在室温下保持打开至少20分钟
    7. 附着的细菌形成生物膜用130μl在乙醇中溶解的1%结晶紫在室温下搅拌5分钟染色。
    8. 然后用200μl无菌PBS洗涤孔5次,无搅拌
    9. 为了干燥它们,将板在室温下放置至少1小时
    10. 加入130μl无水乙醇,将板在室温下不搅拌孵育5分钟
    11. 使用微板振荡器进行10分钟(1000rpm)的高速搅拌
    12.  使用微量培养板分光光度计通过OD 570nm读数估计生物膜形成。
      可以通过使用允许根据M63基本培养基中细菌生长的生物膜形成的标准化的比例OD 570nm/OD 620nm确定比生物膜形成指数。根据以前的出版物,野生型AIEC菌株LF82的特异性生物膜形成指数应该在2和4之间(Martinez-Medina等人,2009; Chassaing和Darfeuille-Michaud,2013) >
  2. 在多聚甲醛固定的I-407 IEC单层上的生物膜形成测定
    肠407(I-407)细胞衍生自人肠道胚胎空肠和回肠组织。将它们在补充有10%(体积/体积)胎牛血清的最低必需培养基中在37℃下保持在含有5%CO 2的气氛中; 1%的非必需氨基酸; 1%的L-谷氨酰胺; 1%的青霉素/链霉素/两性霉素B溶液和1%的维生素混合物
    1. 将单层以4×10 5个细胞/孔接种在24孔组织培养板中的盖玻片上,并在37℃下用5%CO 2孵育20小时。 br />
    2. 然后将单层用1ml PBS洗涤一次,在室温下搅拌下在4%甲醛(稀释于无菌PBS中的储备溶液)中固定15分钟,然后用PBS洗涤4次。
    3. 表达绿色荧光蛋白(GFP)的细菌菌株(Valdivia et al。,1996)如前所述从步骤A1至A2制备,在补充有1mM 的MgSO 4和8g.L -1葡萄糖。
    4. 将1ml该细菌悬浮液施加在固定的I-407细胞单层的表面上,并在30℃下不摇动孵育过夜。
    5. 通过倾析除去液体,使用1ml无菌PBS洗涤孔3次,无需孵育
    6. 然后使用4%甲醛将I-407 /生物膜复合物固定15分钟
    7. 鬼笔环肽 - 四甲基罗丹明异氰酸酯用于显现肌动蛋白,Hoechst 33258用于显现细胞核。为此,将鬼笔环肽 - 四甲基罗丹明异氰酸酯在无菌PBS中以1μg/ml的终浓度稀释,并加入Hoechst 33258,终浓度为1μg/ml。将该溶液加入孔中并在室温下孵育20分钟。孵育后,使用1ml无菌PBS洗涤孔5次,无孵育
    8. 然后从24孔板中取出含有固定和染色的I-407 /生物膜复合物的盖玻片,并使用Vectashield将其固定在载玻片上。
    9. 用共聚焦显微镜检查载玻片。在多聚甲醛固定的I-407 IEC单层上形成生物膜的实例如图1所示
    10. 可以使用计算机程序COMSTAT1(Haydorn等人,2000)分析来自肠上皮细胞I-407单层表面的生物膜形成测定的图像的厚度和/或粗糙度。


      图1.不良生物膜产生器的生物膜形成的共聚焦分析(非致病性大肠杆菌K12菌株C600(A)和强生物膜产生器(粘附侵入性大肠杆菌在PFA固定的I-407肠上皮细胞单层的表面上,跨越> 菌株LF82(B)。表达GFP(绿色),肌动蛋白使用鬼笔环肽-TRITC染色为红色,DNA使用Hoechst.Bars,50μm染色为蓝色。

笔记

  1. 由于对这两种生物膜形成测定观察到高的变化,它们需要至少一式三份进行,包括对照。

食谱

  1. 最低必需培养基,补充有
    10%(vol/vol)胎牛血清 1%的非必需氨基酸
    1%的L-谷氨酰胺 1%青霉素/链霉素/两性霉素B溶液 1%的维生素混合物

致谢

这项研究得到了Ministèrede la Recherche et de la Technologie,the National National de laSantéet de la RechercheMédicaleand theUniversitéd'Auvergne(UMR Inserm 1071),the National National de la Recherche Agronomique(USC INRA 2018)并通过协会F. Aupetit(AFA)的资助。我们感谢CICS平台的共聚焦显微镜。该协议改编自以前发表的论文(Danese等人,2000; Naves等人,2008; Martinez-Medina等人, 2009; Chassaing和Darfeuille-Michaud,2013)。这项工作专用于Arlette Darfeuille-Michaud。

参考文献

  1. Chassaing,B。和Darfeuille-Michaud,A。(2011)。 共生性微生物群和肠病原体在炎症性肠病的发病机制中。胃肠病学 140(6):1720-28。
  2. Chassaing,B。和Darfeuille-Michaud,A。(2013)。 sigmaE通路参与克罗恩病相关的粘附侵入大肠杆菌的生物膜形成。 195(1):76-84
  3. Danese,PN,Pratt,LA,Dove,SL和Kolter,R。(2000)。外膜蛋白,抗原43介导大肠杆菌生物膜内的细胞与细胞的相互作用。 Mol Microbiol 37(2):424-432。
  4. Dirteuille-Michaud,A.,Neut,C.,Barnich,N.,Lederman,E.,Di Martino,P.,Desreumaux,P.,Gambiez,L.,Joly,B.,Cortot, JF(1998)。 克罗恩病患者回肠粘膜中存在粘附的大肠杆菌菌株。 Gastroenterology 115(6):1405-1413。
  5. Martinez-Medina,M.,Naves,P.,Blanco,J.,Aldeguer,X.,Blanco,JE,Blanco,M.,Ponte,C.,Soriano,F.,Darfeuille-Michaud, Gil,LJ(2009)。 生物膜形成作为粘附侵入大肠杆菌的新型表型特征( AIEC)。 BMC Microbiol 9:202。
  6. Heydorn,A.,Nielsen,A.T.,Hentzer,M.,Sternberg,C.,Givskov,M.,Ersboll,B.K.and Molin,S。(2000)。 新型计算机程序COMSTAT对生物膜结构的定量。 微生物学 146(Pt 10):2395-2407。
  7. Naves,P.,del Prado,G.,Huelves,L.,Gracia,M.,Ruiz,V.,Blanco,J.,Dahbi,G.,Blanco,M.,Ponte Mdel,C.and Soriano,F 。(2008)。 毒力因子与大肠杆菌菌株体外生物膜形成的相关性。 Microb Pathog 45(2):86-91
  8. Valdivia,R.H.,Hromockyj,A.E.,Monack,D.,Ramakrishnan,L。和Falkow,S。(1996)。 绿色荧光蛋白(GFP)在宿主 - 病原体相互作用研究中的应用。 Gene 173(1 Spec No):47-52。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chassaing, B. and Darfeuille-Michaud, A. (2013). Adherent-invasive Escherichia coli Biofilm Formation Assays. Bio-protocol 3(23): e982. DOI: 10.21769/BioProtoc.982.
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当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。