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Preparation and Maintenance of Bioexclusion IsoPositive Cage Experiment for Human Fecal Transplantation into Germ-Free Mice
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Short communication: Gut microbial colonization of the mouse colon using faecal transfer was equally effective when
Ditte Olsen Lützhøft1, Lidia Sánchez-Alcoholado2, Peter Tougaard1
1Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark.
Abstract:
In the present study we hypothesized that a higher degree of gut microbiota (GM) transfer and colonization could be reached by rectal inoculation compared to oral inoculation, which is commonly used in mouse studies for GM transfer. We treated C57BL/6NTac Specific Pathogen Free (SPF) mice with antibiotics and subsequently we inoculated these with GM from donor mice of the same strain by either the oral or the rectal inoculation method. 16S rRNA gene sequencing of the colon microbiota showed no difference in microbial community on account of inoculation method as determined by unweighted UniFrac distance metrics in C57BL/6NTac SPF mice. In addition, qPCR analysis on colon tissue revealed no difference in mRNA expression between the inoculation methods. Next, the SPF mice were compared to germ-free (GF)-mice to identify differences in inoculation efficacy. Whether the mice were antibiotic treated SPF or GF clearly influenced GM determined by 16S rRNA gene sequencing where the SPF mice experienced up-regulation of S24-7 (p = .0001) and a decrease in Rikenellaceae (p = .016) compared to GF mice. qPCR analysis on colon tissue revealed up-regulation in mRNA gene expression of Il6, Il10, Reg3g and transcription factor RORγt (Rorc) in GF mice compared to SPF mice on a significant level (p < .05). This gene expression profile is consistent with post colonization development of the intestinal barrier in GF mice.
Insights
Rectal gut microbiota (GM) inoculation showed no difference in colonization compared to oral methods in mice. However, antibiotic-treated specific pathogen-free (SPF) mice differed significantly from germ-free (GF) mice in microbial composition and gene expression.
Area of Science:
- Microbiology
- Gastroenterology
- Immunology
Background:
- Gut microbiota (GM) transfer is crucial for establishing microbial communities.
- Oral inoculation is a common method for GM transfer in mouse models.
- Investigating alternative inoculation methods like rectal administration is important.
Purpose of the Study:
- To compare the efficacy of rectal versus oral gut microbiota inoculation in mice.
- To identify differences in gut microbiota composition and host gene expression between inoculation methods.
- To compare gut microbiota colonization and host response in specific pathogen-free (SPF) versus germ-free (GF) mice.
Main Methods:
- Antibiotic treatment of C57BL/6NTac mice to deplete native microbiota.
- Inoculation with donor gut microbiota via oral or rectal routes.
- 16S rRNA gene sequencing to analyze colon microbiota composition.
- Quantitative PCR (qPCR) to assess host gene expression in colon tissue.
Main Results:
- No significant difference in microbial community composition was observed between oral and rectal inoculation methods in SPF mice.
- No difference in mRNA expression was found between oral and rectal inoculation methods.
- Significant differences in gut microbiota composition and host gene expression were observed between antibiotic-treated SPF mice and GF mice.
- SPF mice showed altered abundance of specific bacterial taxa (S24-7, Rikenellaceae) compared to GF mice.
- GF mice exhibited distinct gene expression profiles (Il6, Il10, Reg3g, Rorc) indicative of developing intestinal barrier function.
Conclusions:
- Rectal inoculation is not superior to oral inoculation for gut microbiota transfer in this mouse model.
- The host's initial microbial status (SPF vs. GF) significantly impacts gut microbiota colonization and host gene expression.
- Germ-free mice demonstrate gene expression patterns consistent with the development of the intestinal barrier post-colonization.
