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Isolation of Commensal Escherichia coli Strains from Feces of Healthy Laboratory Mice or Rats
从健康实验小鼠或大鼠粪便中分离共生大肠埃希杆菌菌株   

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参见作者原研究论文

本实验方案简略版
Applied and Environmental Microbiology
Sep 2017

Abstract

The colonization abundance of commensal E. coli in the gastrointestinal tract of healthy laboratory mice and rats ranges from 104 to 106 CFU/g feces. Although very well characterized, the family that E. coli belongs to has a very homogeneous 16S rRNA gene sequence, making the identification from 16S rRNA sequencing difficult. This protocol provides a procedure of isolating and identifying commensal E. coli strains from a healthy laboratory mouse or rat feces. The method can be applied to isolate commensal E. coli from other laboratory rodent strains.

Keywords: Commensal (共生), Escherichia coli (大肠埃希杆菌), Isolation (分离), Laboratory rodents (实验室啮齿动物)

Background

Escherichia coli is a Gram-negative, facultative anaerobe which constitutes only a minor fraction of the vertebrate gut microbiota, but plays a key role in microbial interaction, immune modulation and metabolic functionalities (Tenaillon et al., 2010). Being one of the best-characterized model microorganisms, commensal E. coli strains have been increasingly studied to unravel the mechanisms through which gut commensal microbes adapt to the unique niche and impact host physiology. However, the high homology among different strains raises difficulties in identification and characterization of commensal E. coli based on a 16S rRNA sequencing approach. Thanks to the development of next-generation sequencing techniques and large-scale analyses of whole genomes, we are able to identify commensal E. coli strains isolated from the gastrointestinal tract of different hosts according to the presence of virulence genes in the genome. In this protocol, we show an approach to isolate and identify commensal E. coli strains from a laboratory mouse or rat using selective culture media and whole genome sequencing. However, it should be noted that the presence of commensal E. coli in laboratory animals depends on the vendor and environmental conditions of the facility.

Materials and Reagents

  1. Gloves and masks (KCWW, Kimberly-Clark, catalog number: 52817 ; Cardinal Health, Insta-Gard, catalog number: AT7511-WE )
  2. 1.5 ml centrifuge tube (sterile) (Fisher Scientific, catalog number: 05-408-129 )
  3. Wide bore tips, 0-200 μl (Corning, Axygen®, catalog number: T-1005-WB-C )
  4. Thin-wall PCR tubes (Fisher Scientific, catalog number: 14-230-225 )
  5. Tips, 0.1-10 μl, 0.1-1 ml (sterile) (Fisher Scientific, catalog numbers: 02-707-474 ; 02-707-480 )
  6. Cell spreader (Fisher Scientific, catalog number: 08-100-11 )
  7. Petri dishes (100 x 15 mm) (Fisher Scientific, catalog number: FB0875713 )
  8. 15 ml conical sterile polypropylene centrifuge tubes (Thermo Fisher Scientific, NuncTM, catalog number: 339650 )
  9. Sterile 0.22 μm filter (Corning, catalog number: 431219 )
  10. 1 ml syringe (BD, catalog number: 309659 )
  11. A healthy NIH Swiss mouse (Harlan Laboratories Inc., Indianapolis, IN) and Sprague-Dawley (SD) rat (Charles River Canada, St. Constant, QC)
  12. 70% ethanol diluted from 100% ethanol (Commercial Alcohols, catalog number: P016EAAN )
  13. Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500500 )
  14. Tris-Acetate-EDTA (TAE) (50x stock) (Fisher Scientific, catalog number: BP13321 )
  15. SYBR Safe DNA gel stain (Thermo Fisher Scientific, InvitrogenTM, catalog number: S33102 )
  16. 6x DNA loading dye (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0611 )
  17. 1 kb Plus DNA ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM1331 )
  18. GeneJET gel extraction and DNA cleanup micro kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: K0832 )
  19. PureLink genomic DNA mini kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: K182001 )
  20. Nextera XT DNA library preparation kit (Illumina, catalog number: FC-131-1096 )
  21. Nextera XT DNA library preparation index kit (Illumina, catalog number: FC-131-1002 )
  22. Nextera XT DNA library preparation kit (Illumina, catalog number: FC-131-1096 )
  23. Nextera XT DNA library preparation index kit (Illumina, catalog number: FC-131-1002 )
  24. QubitTM 1x dsDNA HS assay kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q33230 )
  25. PhiX control kit (Illumina, catalog number: FC-110-3001 )
  26. MiSeq reagent kit V3 (Illumina, catalog number: MS-102-3003 )
  27. Sodium phosphate dibasic (Na2HPO4) (Fisher Scientific, catalog number: S373-500 )
  28. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285-500 )
  29. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-500 )
  30. Potassium chloride (KCl) (Fisher Scientific, catalog number: P217-500 )
  31. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144-500LB )
  32. MacConkey agar (BD, catalog number: 212123 )
  33. Luria-Bertani (LB) broth (Sigma-Aldrich, catalog number: L3022 )
  34. Glycerol (Fisher Scientific, catalog number: BP229-1 )
  35. DNA Taq polymerase with 50 mM MgCl2 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10342020 )
  36. Oligo primers 27F/1492R (Weisburg et al., 1991)
    27F: 5’-AGAGTTTGATCMTGGCTCAG-3’
    1492R: 5’-TACGGYTACCTTGTTACGACTT-3’
  37. Deoxynucleotide triphosphates (dNTPs) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10297-018 )
  38. PCR grade water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9932 )
  39. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: SS266-1 )
  40. 1x phosphate buffered saline (PBS) (pH 7.4) (see Recipes)
  41. MacConkey agar (see Recipes)
  42. LB broth (see Recipes)
  43. Glycerol stock of bacterial isolates (see Recipes)
  44. PCR reaction mix (see Recipes)
  45. 0.1 N NaOH (see Recipes)

Equipment

  1. Forceps (sterilized by autoclave)
  2. Vortex (Fisher Scientific, catalog number: 02215365 )
  3. Pipettes [e.g., P1000 (Eppendorf, catalog number: 3120000062 ), P200 (Eppendorf, catalog number: 3120000054 ), P10 (Eppendorf, catalog number: 3120000020 )]
  4. Thermal cycler (Thermo Fisher Scientific, Applied Biosystems, model: GeneAMP PCR System 9700 )
  5. Microwave (RCA, catalog number: RMW733 )
  6. Gel electrophoresis system (Fisher Scientific, model: FB300 )
  7. UV transilluminator with photo documentation (Azure Biosystems, model: Azure c200 )
  8. Incubator (37 °C) (Thermo Fisher Scientific, Thermo Scientific, model: Model 370 )
  9. Shaking incubator (37 °C) (Eppendorf, New BrunswickTM, model: I26 )
  10. QubitTM 3.0 fluorometer (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q33216 )
  11. QubitTM assay tubes (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32856 )
  12. Illumina MiSeq instrument (Illumina, model: MiSeqTM System, catalog number: SY-410-1003 )
  13. Autoclave (Beta Star Life Science Equipment, model: C2002BS )
  14. -80 °C freezer (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM 900 Series , 989)

Procedure

The protocol for isolating and identifying the gut commensal E. coli strains from laboratory mouse or rat feces includes the steps for fecal collection, bacterial culture and colony characterization (Figure 1).


Figure 1. Workflow scheme for the isolation and identification of commensal E. coli from mouse or rat feces

  1. Fecal collection
    Note: Personal protection equipment including gloves and masks are required.
    1. Put a healthy NIH Swiss mouse/SD rat into a clean cage and wait for defecation.
    2. Use sterile forceps to transfer one fresh fecal pellet into a 1.5 ml centrifuge tube preloaded with 1 ml of 1x PBS.
      Note: Recommended weight of one fresh fecal pellet: mouse, 30-50 mg; rat, 80-100 mg.
    3. Put the tubes with fecal pellet on the ice and immediately transfer them back to the lab to perform serial dilution and plating.

  2. Serial dilution and plating
    Note: Wear gloves and use 70% ethanol as a disinfectant for cleaning the surfaces of working bench.
    1. Homogenize the fecal pellet thoroughly by vortexing for 30 sec at the maximum speed.
    2. Add 900 μl of 1x PBS into four 1.5 ml centrifuge tubes. Perform 1:10 serial dilution from the fecal samples using the wide bore tips.
    3. Use the cell spreaders to plate 100 μl of each dilution onto MacConkey agar plate (10-1 to 10-5).
    4. Incubate the plates aerobically at 37 °C for 16-18 h.

  3. Colony PCR for E. coli identification by Sanger sequencing using 16S rRNA gene primers
    1. MacConkey agar is a selective and differential medium for the isolation of coliform organisms. The potential E. coli colonies appear as red and round shaped colonies due to the capability to ferment lactose (Figure 2). Select potential colonies for the following PCR amplification.


      Figure 2. Colony morphology of E. coli isolated from rat feces after growth on MacConkey agar. The potential E. coli colonies appear as red with a round shape. The colonies pointed with black arrows are considered as negative colonies.

    2. Add 50 μl of PCR reaction mix (see Recipes) into each PCR tube.
    3. Use P10 tips to pick a small amount of the single colony and swirl in the PCR reaction. Mix the colonies and PCR reaction by pipetting up and down, and then discard the tips.
    4. Complete the reaction in a thermal cycler following the PCR program (see Notes).
    5. Prepare 40 ml of 1% agarose gel by dissolving 0.4 g agarose in 1x TAE and heating (microwave). Add 4 μl of SYBR Safe DNA gel stain into the gel and mix thoroughly. Cast the agarose gel in the apparatus and wait until the gel is solidified. Mix 5 μl of PCR reaction with 1 μl 6x DNA loading dye and apply the samples as well as the marker (2 μl, 1 kb Plus DNA ladder) to the gel. Run the electrophoresis for 30 min at 100 V. Take a picture to check the amplification using a UV transilluminator. Compare the size of the PCR product (about 1,465 bp) to the marker (Figure 3).


      Figure 3. Agarose gel electrophoresis image of the 16S rRNA genes amplified from six bacterial isolates. Lane 1 indicates the negative control and the lane 2 to 7 indicate the PCR products amplified the 16S rRNA gene of the selected bacterial isolates. The size of the PCR product is around 1,465 bp.

    6. PCR clean-up is performed using a GeneJET gel extraction and DNA cleanup kit following the manufacturer’s instruction. The remaining PCR mix after the gel electrophoresis is used for this step. With the adjusted concentrations, the cleaned-up samples are further sequenced by Sanger sequencing using the 16S rRNA gene primers.
    7. The 16S rRNA gene sequences are searched against the Ribosomal Database Project (RDP, released 11.4; http://rdp.cme.msu.edu/) and the NCBI nucleotide database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (see Data analysis).
    8. The isolates that share higher than 98% 16S rRNA sequence identity with the type strain of E. coli are selected and cultured in LB broth (see Recipes) overnight in a shaking incubator (37 °C) to make glycerol stocks (25% glycerol, see Recipes) for preservation.

  4. Whole genome sequences and annotation
    1. The selected isolates are cultured overnight in 5 ml LB broth for genomic DNA extraction using a PureLink Genomic DNA Mini Kit following the manufacturer’s instruction. The LB culture from the last Step C8 can be utilized for DNA extraction.
    2. The whole genome sequencing is performed on an Illumina MiSeq platform. The isolated genomic DNA is fragmented to generate libraries using a Nextera XT DNA library preparation kit according to the manufacturer’s instruction.
    3. The concentrations of generated libraries are quantified by a QubitTM 3.0 fluorometer using a QubitTM 1x dsDNA HS assay kit according to the manufacturer’s instruction. The quantified libraries are further normalized to 2 nM and pooled following the protocol of the Nextera XT DNA library preparation kit.
    4. Denature the pooled libraries using 0.1 N NaOH and mix with 5% PhiX genomic DNA as a positive control.
    5. The sequencing of denatured libraries is performed on an Illumina MiSeq instrument with 2 x 300 bp reads generated, using a MiSeq reagent V3 sequencing-by-synthesis kit.
    6. The draft genome is assembled with the SPAdes assembler (Bankevich et al., 2012). Genome assemblies are evaluated by Quality Assessment Tool for Genome Assemblies (QUAST) (Gurevich et al., 2013). The tool of BBMap (Bushnell, 2014) is used to map the raw reads back to the contigs produced by SPAdes to obtain the information about the coverage for contigs. The algorithm of Megablast (Zhang et al., 2000) is applied to blast the contigs against the reference bacterial genomes obtained from NBCI. Rapid Annotations using Subsystems Technology (RAST) (Aziz et al., 2008) is used for genome annotation.
    7. IslandViewer (Dhillon et al., 2015) is used to predict toxin related virulence in the whole genome of the E. coli isolate. The genomes of the isolates submitted to IslandViewer are in the format of GENBANK. The isolates without identified hits of toxin virulence factor (VF)-related genes in the genome are considered to be commensal E. coli isolates.

Data analysis

After colony PCR and Sanger sequencing, the 16S rRNA sequences of bacterial isolates are aligned against the RDP and NCBI nucleotide database. The tools of Seqmatch and Classifier within the RDP database are used for assigning taxonomy. High-quality sequences of the type strains (the size ≥ 1,200 bp) are chosen in the settings of Seqmatch. The ‘16S ribosomal RNA sequences’ database (Bacteria and Archaea) is used as the reference database with default settings when searching against the NCBI database.

Notes

  1. Thermal cycler condition
    Initial denaturation for 10 min at 94 °C
    35 cycles of 30 sec at 94 °C, 30 sec at 58 °C, and 1 min 40 sec at 72 °C
    Final extension for 7 min at 72 °C
    Hold at 4 °C

Recipes

  1. 1x phosphate buffer saline (PBS) (pH 7.4) (1 L)
    10 mM Na2HPO4
    1.8 mM KH2PO4
    137 mM NaCl
    2.7 mM KCl
    Adjust to pH 7.4 with HCl and sterilize by filter or autoclave before use
  2. MacConkey agar
    Suspend 50 g of the powder in 1 L of ddH2O. Mix thoroughly and boil for 1 min to completely dissolve the powder. Autoclave at 121 °C for 15 min. Cool down and dispense approximately 20 ml per Petri dish (100 x 15 mm in diameter). Store at 4 °C for up to a month
  3. LB broth
    Stir to suspend 25 g of the powder in 1 L of ddH2O. Autoclave for 15 min at 121 °C to sterilize. Store at 4 °C for up to a month
  4. Glycerol stock of bacterial isolates (in 25% glycerol)
    1. Prepare 100 ml of 50% (v/v) glycerol
      Glycerol (100%) 50 ml
      Add ddH2O to 100 ml
      Autoclave to sterilize
    2. Make glycerol stock from the broth culture of bacterial isolates
      Use sterile pipet tips, pipet 500 μl of bacterial broth culture into a sterile centrifuge tube
      Add 500 μl of 50% autoclaved glycerol. Mix the solution by vortex and place the tubes into the -80 °C freezer
  5. PCR reaction mix
    5 μl of 10x PCR buffer (1x final concentration)
    0.5 μl of 1 U/μl Taq polymerase
    2 μl of 50 mM MgCl2
    2 μl of 10 μM Oligo primer 27F
    2 μl of 10 μM Oligo primer 1492R
    2 μl of 10 mM dNTP mix
    Add up to 50 μl of total volume with PCR grade water
  6. 0.1 N NaOH solution
    Make a 1:10 dilution of 1 N NaOH (Fisher Scientific) using nuclease-free water. For example: In a sterile 15 ml centrifuge tube, add 9 ml of nuclease-free water into 1 ml of 1 N NaOH solution. Mix the solution by vortex and sterilize by filtration through a 0.22 μm filter with a syringe

Acknowledgments

This research was supported by a Natural Sciences and Engineering Research Council Discovery grant held by B.P.W. T.J. was supported by a Graduate Student Scholarship from Alberta Innovates-Technology Futures, Alberta, Canada. B.P.W. is supported by the Canada Research Chair Program. This protocol was adapted from Ju et al., 2017. The authors have no conflict of interest to declare.

References

  1. Aziz, R. K., Bartels, D., Best, A. A., DeJongh, M., Disz, T., Edwards, R. A., Formsma, K., Gerdes, S., Glass, E. M., Kubal, M., Meyer, F., Olsen, G. J., Olson, R., Osterman, A. L., Overbeek, R. A., McNeil, L. K., Paarmann, D., Paczian, T., Parrello, B., Pusch, G. D., Reich, C., Stevens, R., Vassieva, O., Vonstein, V., Wilke, A. and Zagnitko, O. (2008). The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9: 75.
  2. Bankevich, A., Nurk, S., Antipov, D., Gurevich, A. A., Dvorkin, M., Kulikov, A. S., Lesin, V. M., Nikolenko, S. I., Pham, S., Prjibelski, A. D., Pyshkin, A. V., Sirotkin, A. V., Vyahhi, N., Tesler, G., Alekseyev, M. A. and Pevzner, P. A. (2012). SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19(5): 455-477.
  3. Bushnell, B. (2014). BBMap: A Fast, Accurate, Splice-Aware Aligner. United States.
  4. Dhillon, B. K., Laird, M. R., Shay, J. A., Winsor, G. L., Lo, R., Nizam, F., Pereira, S. K., Waglechner, N., McArthur, A. G., Langille, M. G. and Brinkman, F. S. (2015). IslandViewer 3: more flexible, interactive genomic island discovery, visualization and analysis. Nucleic Acids Res 43(W1): W104-108.
  5. Gurevich, A., Saveliev, V., Vyahhi, N. and Tesler, G. (2013). QUAST: quality assessment tool for genome assemblies. Bioinformatics 29(8): 1072-1075.
  6. Ju, T., Shoblak, Y., Gao, Y., Yang, K., Fouhse, J., Finlay, B. B., So, Y. W., Stothard, P. and Willing, B. P. (2017). Initial gut microbial composition as a key factor driving host response to antibiotic treatment, as exemplified by the presence or absence of commensal Escherichia coli. Appl Environ Microbiol 83.
  7. Tenaillon, O., Skurnik, D., Picard, B. and Denamur, E. (2010). The population genetics of commensal Escherichia coli. Nat Rev Microbiol 8(3): 207-217.
  8. Weisburg, W. G., Barns, S. M., Pelletier, D. A. and Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173(2): 697-703.
  9. Zhang, Z., Schwartz, S., Wagner, L. and Miller, W. (2000). A greedy algorithm for aligning DNA sequences. J Comput Biol 7(1-2): 203-214.

简介

共生E的殖民丰度。 大肠杆菌在健康实验小鼠和大鼠的胃肠道中的范围为10 4至10 6 CFU / g粪便。 虽然描述得非常好,但那个家族就是这样的。 大肠杆菌属于具有非常均一的16S rRNA基因序列,使得从16S rRNA测序鉴定困难。 该协议提供了分离和识别共生E的程序。 来自健康实验室小鼠或大鼠粪便的大肠杆菌菌株。 该方法可以应用于隔离共生电子。 来自其他实验室啮齿类动物的大肠杆菌。

【背景】大肠杆菌是革兰氏阴性兼性厌氧菌,其仅构成脊椎动物肠道微生物群的一小部分,但在微生物相互作用,免疫调节和代谢功能中起关键作用(Tenaillon等人。,2010)。作为最好的模式微生物之一,共生E。已经越来越多地研究大肠杆菌菌株以揭示肠道共生微生物适应独特生态位并影响宿主生理机制。然而,不同菌株之间的高度同源性在共生E的鉴定和表征上提出了困难。基于16S rRNA测序方法的大肠杆菌。由于新一代测序技术的发展和全基因组的大规模分析,我们能够识别共生E。根据基因组中毒力基因的存在,分离自不同宿主的胃肠道的大肠杆菌菌株。在这个协议中,我们展示了一种分离和识别共生E的方法。使用选择性培养基和全基因组测序从实验室小鼠或大鼠获得大肠杆菌菌株。但是,应该指出的是,共生E的存在。大肠杆菌在实验室动物中取决于设施的供应商和环境条件。

关键字:共生, 大肠埃希杆菌, 分离, 实验室啮齿动物

材料和试剂

  1. 手套和口罩(KCWW,Kimberly-Clark,目录号:52817; Cardinal Health,Insta-Gard,目录号:AT7511-WE)
  2. 1.5 ml离心管(无菌)(Fisher Scientific,目录号:05-408-129)
  3. 宽口径吸头,0-200μl(Corning,Axygen ®,目录号:T-1005-WB-C)
  4. 薄壁PCR管(Fisher Scientific,目录号:14-230-225)
  5. 尖端,0.1-10μl,0.1-1ml(无菌)(Fisher Scientific,目录号:02-707-474; 02-707-480)
  6. 细胞撒布机(Fisher Scientific,目录号:08-100-11)
  7. 培养皿(100 x 15 mm)(Fisher Scientific,目录号:FB0875713)
  8. 15ml锥形无菌聚丙烯离心管(Thermo Fisher Scientific,Nunc TM,目录号:339650)
  9. 无菌0.22μm过滤器(Corning,目录号:431219)
  10. 1毫升注射器(BD,目录号:309659)

  11. 健康的NIH Swiss小鼠(Harlan Laboratories Inc.,Indianapolis,IN)和Sprague-Dawley(SD)大鼠(Charles River Canada,St. Constant,QC)
  12. 用100%乙醇稀释的70%乙醇(商业醇,目录号:P016EAAN)
  13. 琼脂糖(Thermo Fisher Scientific,Invitrogen TM,目录号:16500500)
  14. Tris-Acetate-EDTA(TAE)(50x储备液)(Fisher Scientific,目录号:BP13321)
  15. SYBR Safe DNA凝胶染色(Thermo Fisher Scientific,Invitrogen TM,目录号:S33102)
  16. 6x DNA上样染料(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0611)
  17. 1 kb Plus DNA梯(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:SM1331)
  18. GeneJET凝胶提取和DNA清除微量试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:K0832)
  19. PureLink基因组DNA微型试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:K182001)
  20. Nextera XT DNA文库制备试剂盒(Illumina,目录号:FC-131-1096)
  21. Nextera XT DNA文库制备指数试剂盒(Illumina,目录号:FC-131-1002)
  22. Nextera XT DNA文库制备试剂盒(Illumina,目录号:FC-131-1096)
  23. Nextera XT DNA文库制备指数试剂盒(Illumina,目录号:FC-131-1002)
  24. Qubit TM 1x dsDNA HS分析试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q33230)
  25. PhiX控制组件(Illumina,目录号:FC-110-3001)
  26. MiSeq试剂盒V3(Illumina,目录号:MS-102-3003)
  27. 磷酸二氢钠(Na 2 HPO 4)(Fisher Scientific,目录号:S373-500)
  28. 磷酸二氢钾(KH 2 PO 4)(Fisher Scientific,目录号:P285-500)
  29. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-500)
  30. 氯化钾(KCl)(Fisher Scientific,目录号:P217-500)
  31. 盐酸(HCl)(Fisher Scientific,目录号:A144-500LB)
  32. MacConkey琼脂(BD,目录号:212123)
  33. Luria-Bertani(LB)肉汤(Sigma-Aldrich,目录号:L3022)
  34. 甘油(Fisher Scientific,目录号:BP229-1)
  35. DNA Taq聚合酶与50mM MgCl2(Thermo Fisher Scientific,Invitrogen TM,目录号:10342020)。
  36. Oligo引物27F / 1492R(Weisburg等人,1991)
    27F:5'-AGAGTTTGATCMTGGCTCAG-3'
    1492R:5'-TACGGYTACCTTGTTACGACTT-3'
  37. 三磷酸脱氧核苷酸(dNTPs)(Thermo Fisher Scientific,Invitrogen TM,目录号:10297-018)
  38. PCR级别的水(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9932)
  39. 氢氧化钠(NaOH)(Fisher Scientific,目录号:SS266-1)
  40. 1x磷酸盐缓冲盐水(PBS)(pH 7.4)(见食谱)
  41. MacConkey琼脂(见食谱)
  42. LB肉汤(请参阅食谱)
  43. 细菌分离物的甘油库存(见食谱)
  44. PCR反应混合物(见食谱)
  45. 0.1 N NaOH(见食谱)

设备

  1. 镊子(用高压灭菌器灭菌)
  2. 涡旋(Fisher Scientific,目录号:02215365)
  3. 移液器[例如,P1000(Eppendorf,目录号:3120000062),P200(Eppendorf,目录号:3120000054),P10(Eppendorf,目录号:3120000020)]
  4. 热循环仪(Thermo Fisher Scientific,Applied Biosystems,型号:GeneAMP PCR System 9700)
  5. 微波炉(RCA,目录号:RMW733)
  6. 凝胶电泳系统(Fisher Scientific,型号:FB300)
  7. 具有照片文档的UV透照器(Azure Biosystems,型号:Azure c200)
  8. 培养箱(37°C)(Thermo Fisher Scientific,Thermo Scientific,型号:370型)
  9. 摇动培养箱(37°C)(Eppendorf,New Brunswick TM,型号:I26)
  10. Qubit TM 3.0荧光计(Thermo Fisher Scientific,Invitrogen TM,目录号:Q33216)
  11. Qubit TM测定管(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32856)。
  12. Illumina MiSeq仪器(Illumina,型号:MiSeq TM系统,目录号:SY-410-1003)
  13. 高压灭菌器(Beta Star生命科学设备,型号:C2002BS)
  14. -80°C冷冻箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM 900 Series,989)

程序

用于分离和识别肠共生的协议。来自实验室小鼠或大鼠粪便的大肠杆菌菌株包括粪便收集,细菌培养和菌落表征的步骤(图1)。


图1.从小鼠或大鼠粪便中分离和鉴定共生大肠杆菌的工作流程图

  1. 粪便收集
    注意:个人防护装备包括手套和口罩是必需的。
    1. 将健康的NIH Swiss鼠标/ SD大鼠放入干净的笼子并等待排便。
    2. 使用无菌镊子将一个新鲜的粪便颗粒转移到预先装有1ml 1x PBS的1.5ml离心管中。
      注:推荐新鲜粪便颗粒的重量:小鼠,30-50毫克;大鼠,80-100毫克。
    3. 将带有粪便颗粒的试管放在冰上,立即将它们转移回实验室进行连续稀释和电镀。

  2. 连续稀释和电镀
    注意:戴上手套并使用70%乙醇作为消毒剂清洁工作台表面。
    1. 通过以最大速度涡旋30秒来彻底均化粪便颗粒。
    2. 将900μl1x PBS加入四个1.5 ml离心管中。
      使用大口径吸头对粪便样品进行1:10系列稀释。
    3. 使用细胞涂布器将100μl的每种稀释液涂布到MacConkey琼脂平板(10 -1 -1至10 -5 -5)上。
    4. 在37℃有氧培养板16-18小时。

  3. 用于E的菌落PCR。通过使用16S rRNA基因引物的Sanger测序鉴定大肠杆菌
    1. 麦康凯琼脂是分离大肠菌群的选择性和差异培养基。潜力 E。由于能够发酵乳糖(图2),大肠杆菌菌落显示为红色和圆形菌落。选择潜在的菌落进行下列PCR扩增。


      图2. E的菌落形态。在MacConkey琼脂上生长后,从大鼠粪便中分离出来的大肠杆菌。大肠杆菌菌落呈红色,呈圆形。
      带有黑色箭头的殖民地被视为负面殖民地。


    2. 每个PCR管中加入50μlPCR反应混合物(见配方)。
    3. 使用P10提示在PCR反应中挑选少量单菌落和漩涡。通过上下移液来混合菌落和PCR反应,然后丢弃提示。

    4. 按照PCR程序完成热循环仪中的反应(见注)。
    5. 通过在1x TAE中溶解0.4g琼脂糖并加热(微波)来制备40ml的1%琼脂糖凝胶。将4μlSYBR Safe DNA凝胶染色液加入凝胶中并充分混匀。在装置中浇铸琼脂糖凝胶并等待凝胶凝固。将5μlPCR反应液与1μl6x DNA上样染料混合,并将样品以及标记(2μl,1 kb Plus DNA ladder)应用于凝胶。在100 V下运行电泳30分钟。拍摄照片,使用紫外透射仪检查扩增情况。
      比较PCR产物的大小(大约1,465bp)与标记物(图3)。


      图3.从六种细菌分离物扩增的16S rRNA基因的琼脂糖凝胶电泳图像。泳道1表示阴性对照,泳道2至7表示PCR产物扩增了所选细菌分离物的16S rRNA基因。 PCR产物的大小约为1465 bp。

    6. 使用GeneJET凝胶提取和DNA清洁试剂盒按照生产商的说明进行PCR清洁。该步骤使用凝胶电泳后的剩余PCR混合物。调整浓度后,使用16S rRNA基因引物通过Sanger测序进一步对清理的样品进行测序。
    7. 对核糖体数据库项目(RDP,发布11.4; http://rdp.cem。)搜索16S rRNA基因序列。 msu.edu/ )和NCBI核苷酸数据库( https:// blast。 ncbi.nlm.nih.gov/Blast.cgi )(见数据分析)。
    8. 选择与大肠杆菌的类型菌株具有高于98%16S rRNA序列同一性的分离物并在LB培养基(参见配方)中在振荡培养箱(37℃)中培养过夜以制备甘油库存(25%甘油,见食谱)保存。

  4. 全基因组序列和注释
    1. 使用PureLink Genomic DNA Mini试剂盒按照生产商的说明将选定的分离物在5ml LB肉汤中培养过夜以进行基因组DNA提取。来自最后一步C8的LB培养物可用于DNA提取。
    2. 全基因组测序是在Illumina MiSeq平台上进行的。根据制造商的说明使用Nextera XT DNA文库制备试剂盒将分离的基因组DNA片段化以产生文库。
    3. 根据制造商的说明,使用Qubit TM 1x dsDNA HS分析试剂盒,通过Qubit TM 3.0荧光计对产生的文库的浓度进行定量。将量化的文库进一步标准化至2nM,并按照Nextera XT DNA文库制备试剂盒的方案汇集。
    4. 使用0.1N NaOH将合并的文库变性并与5%PhiX基因组DNA混合作为阳性对照。
    5. 使用MiSeq试剂V3测序合成试剂盒在Illumina MiSeq仪器上进行变性文库的测序,产生2×300bp读数。
    6. 基因组草图与SPAdes汇编程序汇编在一起(Bankevich et al。,2012)。基因组组装评估由基因组组装质量评估工具(QUAST)评估(Gurevich et al。,2013)。 BBMap的工具(Bushnell,2014)用于将原始回读映射回由SPAdes生成的重叠群,以获得有关重叠群覆盖率的信息。应用Megablast算法(Zhang等人,2000)将爆发重叠群对从NBCI获得的参考细菌基因组进行爆破。使用子系统技术的快速注释(RAST)(Aziz et。,2008)用于基因组注释。
    7. IslandViewer(Dhillon等人,2015)用于预测E全基因组中的毒素相关毒力。 c。oli分离物。提交给IslandViewer的分离株的基因组格式为GENBANK。没有鉴定到基因组中毒素毒力因子(VF)相关基因命名的分离株被认为是共生的大肠杆菌分离株。

数据分析

经菌落PCR和Sanger测序后,将细菌分离株的16S rRNA序列与RDP和NCBI核苷酸数据库进行比对。 RDP数据库中的Seqmatch和分类器工具用于分配分类。在Seqmatch的环境中选择高品质的类型菌株序列(大小≥1,200 bp)。当对NCBI数据库进行检索时,“16S核糖体RNA序列”数据库(细菌和古细菌)被用作参考数据库,其默认设置。

笔记

  1. 热循环仪条件
    94°C初始变性10分钟

    35个循环,94℃30秒,58℃30秒,72℃1分40秒 在72°C下最后延伸7分钟
    保持在4°C

食谱

  1. 1x磷酸盐缓冲盐水(PBS)(pH 7.4)(1 L)
    10mM Na 2 HPO 4 4/2 1.8mM KH 2 PO 4 4/2 137mM NaCl
    2.7 mM KCl
    用HCl调节pH值至7.4,使用前用过滤器或高压灭菌器灭菌
  2. MacConkey琼脂
    将50g粉末悬浮在1L ddH 2 O中。充分混合并煮沸1分钟以完全溶解粉末。在121°C高压灭菌15分钟。每个培养皿冷却并分配约20ml(直径100×15mm)。
    在4°C储存长达一个月
  3. LB肉汤
    搅拌以将25g粉末悬浮在1L ddH 2 O中。在121°C高压灭菌15分钟以灭菌。
    在4°C储存长达一个月
  4. 细菌分离株的甘油原种(在25%甘油中)
    1. 准备100毫升的50%(v / v)甘油
      甘油(100%)50毫升
      将ddH <2> O加入100毫升
      高压灭菌器消毒
    2. 从细菌分离物的肉汤培养物中制备甘油原液
      使用无菌移液枪头,将500μl细菌肉汤培养液移入无菌离心管中。
      加入500μl50%高压灭菌甘油。通过涡旋混合溶液并将管放入-80°C冰箱中。
  5. PCR反应混合物
    5μl10x PCR缓冲液(1x终浓度)
    0.5μl1 U /μlTaq聚合酶
    2μl的50mM MgCl 2 /
    2μl的10μMOligo底漆27F
    2μl10μMOligo底漆1492R
    2μl的10mM dNTP混合物

    用PCR级别的水加总量达50μl
  6. 0.1 N NaOH溶液
    用无核酸酶的水将1N NaOH(Fisher Scientific)1:10稀释。例如:在无菌15 ml离心管中,将9 ml无核酸酶的水加入1 ml 1 N NaOH溶液中。通过涡旋混合溶液并通过使用注射器通过0.22μm过滤器过滤进行灭菌

致谢

这项研究得到了B.P.W所持有的自然科学和工程研究委员会发现赠款的支持。 T.J.由加拿大阿尔伯塔省Alberta Innovates-Technology Futures的研究生奖学金提供支持。 B.P.W.由加拿大研究主席计划提供支持。该协议摘自Ju et al。,2017年。作者没有利益冲突声明。

参考

  1. Aziz,RK,Bartels,D.,Best,AA,DeJongh,M.,Disz,T.,Edwards,RA,Formsma,K.,Gerdes,S.,Glass,EM,Kubal,M.,Meyer,F. ,Olsen,GJ,Olson,R.,Osterman,AL,Overbeek,RA,McNeil,LK,Paarmann,D.,Paczian,T.,Parrello,B.,Pusch,GD,Reich,C.,Stevens,R. ,Vassieva,O.,Vonstein,V.,Wilke,A。和Zagnitko,O。(2008)。 RAST服务器:使用子系统技术的快速注释 BMC Genomics 9:75.
  2. Bankevich,A.,Nurk,S.,Antipov,D.,Gurevich,AA,Dvorkin,M.,Kulikov,AS,Lesin,VM,Nikolenko,SI,Pham,S.,Prjibelski,AD,Pyshkin,AV,Sirotkin ,AV,Vyahhi,N.,Tesler,G.,Alekseyev,MA和Pevzner,PA(2012)。 SPAdes:一种新的基因组组装算法及其在单细胞测序中的应用 < J Comput Biol 19(5):455-477。
  3. Bushnell,B。(2014)。 BBMap简短对齐机构联合染色体研究所能源部。
  4. Dhillon,B. K.,Laird,M. R.,Shay,J. A.,Winsor,G.L.,Lo,R.,Nizam,F.,Pereira,S.K。,Waglechner,N.,McArthur,A.G.,Langille,M.G.and Brinkman,F.S。(2015)。 IslandViewer 3:更灵活,互动的基因组岛发现,可视化和分析 核酸研究43(W1):W104-108。
  5. Gurevich,A.,Saveliev,V.,Vyahhi,N.和Tesler,G。(2013)。 QUAST:用于基因组组装的质量评估工具 生物信息学 29(8):1072-1075。
  6. Ju,T.,Shoblak,Y.,Gao,Y.,Yang,K.,Fouhse,J.,Finlay,B.B.,So,Y.W。,Stothard,P。和Willing,B.P.(2017)。 初始肠道微生物组成是驱动宿主对抗生素反应的关键因素治疗,例如共生大肠杆菌的存在或不存在。应用环境微生物 83。
  7. Tenaillon,O.,Skurnik,D.,Picard,B。和Denamur,E。(2010)。 共生大肠杆菌的群体遗传学。 Nat Rev Microbiol 8(3):207-217。
  8. Weisburg,W.G.,Barns,S.M.,Pelletier,D.A。和Lane,D.J。(1991)。 用于系统发育研究的16S核糖体DNA扩增。 Bacteriol 173(2):697-703。
  9. Zhang,Z.,Schwartz,S.,Wagner,L.和Miller,W。(2000)。 一种用于比对DNA序列的贪婪算法。 J Comput Biol 7(1-2):203-214。
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引用:Ju, T. and Willing, B. P. (2018). Isolation of Commensal Escherichia coli Strains from Feces of Healthy Laboratory Mice or Rats. Bio-protocol 8(6): e2780. DOI: 10.21769/BioProtoc.2780.
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