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, Escherichia coli, Isolation, Laboratory rodents
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
Equipment
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
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
Recipes
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
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