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

Tingting  Ju; Benjamin P. Willing

doi:10.21769/BioProtoc.2780

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Peer-reviewed

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

Tingting Ju

Benjamin P. Willing email

发布: 2018年03月20日第8卷第6期 DOI: 10.21769/BioProtoc.2780 浏览次数: 9919

评审: David CisnerosWolf Dieter RötherAnonymous reviewer(s)

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Cover of Applied and Environmental Microbiology, featuring study using the protocol.

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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

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

Equipment

Forceps (sterilized by autoclave)
Vortex (Fisher Scientific, catalog number: 02215365 )
Pipettes [e.g., P1000 (Eppendorf, catalog number: 3120000062 ), P200 (Eppendorf, catalog number: 3120000054 ), P10 (Eppendorf, catalog number: 3120000020 )]
Thermal cycler (Thermo Fisher Scientific, Applied Biosystems, model: GeneAMP PCR System 9700 )
Microwave (RCA, catalog number: RMW733 )
Gel electrophoresis system (Fisher Scientific, model: FB300 )
UV transilluminator with photo documentation (Azure Biosystems, model: Azure c200 )
Incubator (37 °C) (Thermo Fisher Scientific, Thermo Scientific, model: Model 370 )
Shaking incubator (37 °C) (Eppendorf, New Brunswick^TM, model: I26 )
Qubit^TM 3.0 fluorometer (Thermo Fisher Scientific, Invitrogen^TM, catalog number: Q33216 )
Qubit^TM assay tubes (Thermo Fisher Scientific, Invitrogen^TM, catalog number: Q32856 )
Illumina MiSeq instrument (Illumina, model: MiSeq^TM System, catalog number: SY-410-1003 )
Autoclave (Beta Star Life Science Equipment, model: C2002BS )
-80 °C freezer (Thermo Fisher Scientific, Thermo Scientific^TM, model: Forma^TM 900 Series , 989)

Procedure

English

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如何引用

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.