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. 2014 Mar-Apr;5(2):183-91.
doi: 10.4161/gmic.28403. Epub 2014 Mar 5.

Broad scope method for creating humanized animal models for animal health and disease research through antibiotic treatment and human fecal transfer

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Broad scope method for creating humanized animal models for animal health and disease research through antibiotic treatment and human fecal transfer

Korry J Hintze et al. Gut Microbes. 2014 Mar-Apr.

Abstract

Traditionally, mouse humanization studies have used human fecal transfer to germ-free animals. This practice requires gnotobiotic facilities and is restricted to gnotobiotic mouse lines, which limits humanized mouse research. We have developed a generalizable method to humanize non germ-free mice using antibiotic treatment and human fecal transfer. The method involves depleting resident intestinal microbiota with broad-spectrum antibiotics, introducing human microbiota from frozen fecal samples by weekly gavage, and maintaining mice in HEPA-filtered microisolator cages. Pyrosequencing cecal microbiota 16S rRNA genes showed that recipient mice adopt a humanized microbiota profile analogous to their human donors, and distinct from mice treated with only antibiotics (no fecal transfer) or untreated control mice. In the humanized mice, 75% of the sequence mass was observed in their respective human donor and conversely, 68% of the donor sequence mass was recovered in the recipient mice. Principal component analyses of GC- and HPLC-separated cecal metabolites were performed to determine effects of transplanted microbiota on the metabolome. Cecal metabolite profiles of mice treated with only antibiotics (no fecal transfer) and control mice were dissimilar from each other and from humanized mice. Metabolite profiles for mice humanized from different donor samples clustered near each other, yet were sufficiently distinct that separate clusters were apparent for each donor. Also, cecal concentrations of 57 metabolites were significantly different between humanization treatments. These data demonstrate that our protocol can be used to humanize non germ-free mice and is sufficiently robust to generate metabolomic differences between mice humanized from different human donors.

Keywords: antibiotics; cecal metabolites; fecal transfer; gut microbiota; humanization; mice.

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Figures

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Figure 1. Effect of antibiotic treatment and fecal transfer with human microbiota on (A) final body weight, (B) fasting, whole blood glucose, (C) cecal content weight, and (D) empty cecum weight. Experimental groups are as follows: Control, no antibiotic treatment or fecal transplant; AB-Only, mice dosed with antibiotics but no fecal transfer; D1-Weekly, mice gavaged weekly with microbiota from donor 1; D1-Single, mice gavaged once with microbiota from donor 1; D2-Weekly, mice gavaged weekly with microbiota from donor 2; D2-Single, mice gavaged once with microbiota from donor 2. Values shown are average ± SD (n = 4 to 10 mice). Groups indicated with different labels are significantly different (P < 0.05) as determined by one-way ANOVA with Tukey’s post-hoc test for multiple comparisons.
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Figure 2. Weighted Unifrac tree of cecal microbiota. Branches were colored according to the sample material as follows: orange, control mice no antibiotics or fecal transfer; red, antibiotic-only mice, no fecal transfer; dark green, Donor 1 original source bacteria; light green, mice dosed singly with bacteria from Donor 1; medium green, mice dosed weekly with bacteria from donor 1; dark blue, Donor 2 original source bacteria; light blue, mice dosed singly with bacteria from Donor 2; medium blue, mice dosed weekly with bacteria from Donor 2.
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Figure 3. Time-course of changes in fecal microbiota. Data are based on terminal restriction fragment length polymorphism analysis of fecal bacterial collected at each time point. Values shown are the mean Euclidean distance from the immediate post antibiotic treatment sample derived from principle coordinate vectors 1, 2, and 3 of the Pearson population distance matrix. Abbreviations are D1, Donor 1; D2, Donor 2; and AB, antibiotic.
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Figure 4. Bacterial lineages in (A) Donor 1 found in Donor 1 recipient mice gavaged weekly and (B) Donor 2 found in Donor 2 recipient mice gavaged weekly. prokMSA IDs are colored according to bacteria families, as indicated in the figure legend, while each branch is colored according to its detection in the recipient mouse, either not detected (gray) or positively detected (red).
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Figure 5. Source of bacterial lineages found in (A) Donor 1 recipient mice gavaged weekly and (B) Donor 2 recipient mice gavaged weekly. prokMSA IDs are colored according to bacteria families, as indicated in the figure legend, while each branch is colored according to its source, as follows: red, detected in the donor only; pink, detected in both the donor and recipient mouse; blue, detected in control mice only; and gray, detected in neither control or donor.
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Figure 6. Metabolomic profile of cecal metabolites by partial least square discriminate analysis (PLS-DA) of (A) GC-MS and (B) HILIC-MS analytes. Abbreviations are as follows: Con, control mice (no antibiotic treatment or fecal transfer); AB, antibiotic treatment only; D1, mice dosed weekly with bacteria from Donor 1; and D2, mice dosed weekly with bacteria from Donor 2.
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Figure 7. Effect of experimental treatments on the cecal metabolome. The numbers shown in the figure denote the number of significant, differentially expressed metabolites between treatments determined by either GC-MS or HILIC-MS. Listings of these metabolites and pairwise comparisons among sample groups are provided in Tables S1–S8.

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