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Reproducible Colonization of Germ-Free Mice With the Oligo-Mouse-Microbiota in Different Animal Facilities

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Reproducible Colonization of Germ-Free Mice With the Oligo-Mouse-Microbiota in Different Animal Facilities

Claudia Eberl et al. Front Microbiol.

Abstract

The Oligo-Mouse-Microbiota (OMM12) is a recently developed synthetic bacterial community for functional microbiome research in mouse models (Brugiroux et al., 2016). To date, the OMM12 model has been established in several germ-free mouse facilities world-wide and is employed to address a growing variety of research questions related to infection biology, mucosal immunology, microbial ecology and host-microbiome metabolic cross-talk. The OMM12 consists of 12 sequenced and publically available strains isolated from mice, representing five bacterial phyla that are naturally abundant in the murine gastrointestinal tract (Lagkouvardos et al., 2016). Under germ-free conditions, the OMM12 colonizes mice stably over multiple generations. Here, we investigated whether stably colonized OMM12 mouse lines could be reproducibly established in different animal facilities. Germ-free C57Bl/6J mice were inoculated with a frozen mixture of the OMM12 strains. Within 2 weeks after application, the OMM12 community reached the same stable composition in all facilities, as determined by fecal microbiome analysis. We show that a second application of the OMM12 strains after 72 h leads to a more stable community composition than a single application. The availability of such protocols for reliable de novo generation of gnotobiotic rodents will certainly contribute to increasing experimental reproducibility in biomedical research.

Keywords: 3R; Oligo-MM12; defined bacterial consortia; gnotobiology; isobiotic mice; minimal microbiome; sDMDMm2; syncom.

Figures

FIGURE 1
FIGURE 1
Dynamics of fecal community composition after inoculation of OMM12 colonization in germ-free mice. (A) Experimental scheme. Germ-free mice were inoculated with the OMM12 mixture and kept in a germ-free isolator in facility 4. Total number of mice and collection time points of fecal samples are indicated. (B) Relative abundance of fecal microbiota composition at the indicated time points. Abundance of individual strains is shown as relative abundance and expressed as% of cumulative 16S rRNA gene copy numbers of all OMM12 strains. One bar corresponds to one mouse. (C) PCoA based on the distance matrix of Bray–Curtis dissimilarity of relative OMM12 abundance profiles shows the effect of time after inoculation. Points are colored by time (days) after inoculation. Samples taken from two mice are connected to visualize their trajectory during time.
FIGURE 2
FIGURE 2
Absolute abundance of individual OMM12 strains in time course analysis of OMM12 colonization in germ-free mice. Germ-free mice were inoculated with the OMM12 mixture and kept in a germ-free isolator. Fecal samples were collected at different time points for microbiota analysis. Absolute abundance of each strain was determined by a strain-specific qPCR assay and is plotted as 16S rRNA gene copy numbers of the individual strains per μl of extracted gDNA: (A) Lactobacillus reuteri I49, (B) Enterococcus faecalis KB1, (C) Blautia coccoides YL58, (D) Clostridium innocuum I46, (E) Flavonifractor plautii YL31, (F) Clostridium clostridioforme YL32, (G) Acutalibacter muris KB18, (H) Bacteroides caecimuris I48, (I) Muribaculum intestinale YL27, (J) Bifidobacterium longum subsp. animalis YL2, (K) Akkermansia muciniphila YL44, (L) Turicimonas muris YL45. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparison test (p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Green symbols indicate samples collected <20 days post inoculation. Dotted lines indicate detection limits.
FIGURE 3
FIGURE 3
Colonization dynamics of OMM12 mice in four different germ-free facilities after single-dose inoculation. (A) Experimental scheme. Germ-free C57BL/6J mice were inoculated with the OMM12 mixtures and kept in germ-free isolators or gnotocages at four different animal facilities (1-1, 2, 3, 4-1); the number of mice and collection time points of fecal samples are indicated. (B) Fecal microbiota composition at the different time points, displayed as relative abundance and expressed as the fraction of cumulated 16S rRNA gene copy numbers. One bar corresponds to one mouse. (C) PCoA based on Bray–Curtis dissimilarity (relative abundances) between samples obtained from mice in different facilities. Points are colored by facility.
FIGURE 4
FIGURE 4
Colonization dynamics of OMM12 mice in four different germ-free facilities after single-dose inoculation: absolute abundance of individual OMM12 strains. Absolute abundance of each strain was determined using a strain-specific qPCR assay for the experiment described in Figure 3, Data are plotted as 16S rRNA gene copy numbers of the individual strains per μl of extracted gDNA: (A) Lactobacillus reuteri I49, (B) Enterococcus faecalis KB1, (C) Blautia coccoides YL58, (D) Clostridium innocuum I46, (E) Flavonifractor plautii YL31, (F) Clostridium clostridioforme YL32, (G) Acutalibacter muris KB18, (H) Bacteroides caecimuris I48, (I) Muribaculum intestinale YL27, (J) Bifidobacterium longum subsp. animalis YL2, (K) Akkermansia muciniphila YL44, (L) Turicimonas muris YL45. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparison test (p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Green points indicate samples collected <20 days post inoculation.
FIGURE 5
FIGURE 5
Colonization dynamics of OMM12 mice in four different germ-free facilities after double-dose inoculation reveals high reproducibility. (A) Experimental scheme. Germ-free C57BL/6 mice were inoculated twice with the OMM12 mixtures and kept in germ-free isolators or gnotocages at different facilities (1-2, 5, 4-2, 4-3). In case of facility “1” and “4,” inoculations were done on several independent occasions. The number of mice and collection time points of fecal samples are indicated. (B) Fecal microbiota composition at the different time points. Microbiota composition is shown as relative abundance and expressed as the fraction of cumulated 16S rRNA gene copy numbers. One bar corresponds to one mouse. (C) PCoA based on the distance matrix of Bray–Curtis dissimilarity of relative OMM12 abundance profiles shows samples obtained from mice in different facilities. Points are colored by facility.
FIGURE 6
FIGURE 6
Absolute abundance of OMM12 strains in different germ-free facilities after double-dose inoculation. Absolute abundance of each strain was determined using a strain-specific qPCR assay for the experiment described in Figure 5, Data are plotted as 16S rRNA gene copy numbers of the individual strains per μl of extracted gDNA: (A) Lactobacillus reuteri I49, (B) Enterococcus faecalis KB1, (C) Blautia coccoides YL58, (D) Clostridium innocuum I46, (E) Flavonifractor plautii YL31, (F) Clostridium clostridioforme YL32, (G) Acutalibacter muris KB18, (H) Bacteroides caecimuris I48, (I) Muribaculum intestinale YL27, (J) Bifidobacterium longum subsp. animalis YL2, (K) Akkermansia muciniphila YL44, (L) Turicimonas muris YL45. Statistical analysis was performed using Kruskal–Wallis test with Dunn’s multiple comparison test (p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Green color indicate samples collected <20 days post inoculation.
FIGURE 7
FIGURE 7
OMM12 community profiles within and between different facilities for the two trials. The mean of pairwise BC dissimilarity values of the community profiles of mice housed in the same facility (within facility analysis) was plotted against the mean pairwise BC dissimilarity values of the community profile of mice located in different facilities for single-dose inoculation trial for (A) Single-dose inoculation and (B) double-dose inoculation trial. For single-dose inoculation, the mean BC dissimilarity is 0.12 ± 0.04 (mean ± SD) and 0.18 ± 0.04, within and between the facilities, respectively (p-value = 0.07, paired two-sided t-test). For double-dose inoculation, the mean BC dissimilarity is 0.16 ± 0.06 and 0.19 ± 0.03 for within and between the facilities, respectively (p-value = 0.25; paired two-sided t-test).

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References

    1. Anderson M. J., Willis T. J. (2003). Canonical analysis of principal coordinates: a useful method of constrained ordination for ecology. Ecology 84 511–525. 10.1890/0012-9658(2003)084[0511:CAOPCA]2.0.CO;2 - DOI
    1. Bangsgaard Bendtsen K. M., Krych L., Sorensen D. B., Pang W., Nielsen D. S., Josefsen K., et al. (2012). Gut microbiota composition is correlated to grid floor induced stress and behavior in the BALB/c mouse. PLoS One 7:e46231. 10.1371/journal.pone.0046231 - DOI - PMC - PubMed
    1. Barroso-Batista J., Sousa A., Lourenco M., Bergman M. L., Sobral D., Demengeot J., et al. (2014). The first steps of adaptation of Escherichia coli to the gut are dominated by soft sweeps. PLoS Genet. 10:e1004182. 10.1371/journal.pgen.1004182 - DOI - PMC - PubMed
    1. Becker N., Kunath J., Loh G., Blaut M. (2011). Human intestinal microbiota: characterization of a simplified and stable gnotobiotic rat model. Gut Microbes 2 25–33. 10.4161/gmic.2.1.14651 - DOI - PubMed
    1. Bergstrom J. H., Berg K. A., Rodriguez-Pineiro A. M., Stecher B., Johansson M. E., Hansson G. C. (2014). AGR2, an endoplasmic reticulum protein, is secreted into the gastrointestinal mucus. PLoS One 9:e104186. 10.1371/journal.pone.0104186 - DOI - PMC - PubMed

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