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Variation in Taxonomic Composition of the Fecal Microbiota in an Inbred Mouse Strain across Individuals and Time.

Hoy YE, Bik EM, Lawley TD, Holmes SP, Monack DM, Theriot JA, Relman DA - PLoS ONE (2015)

Bottom Line: However, the relative contributions of these factors in adult animals, as well as variation with time in a variety of important settings, are still not fully understood.Our findings suggest that the microbiota of controlled populations of inbred laboratory animals may not be as uniform as previously thought, that animal rearing and handling may account for some variation, and that as yet unidentified factors may explain additional components of variation in the composition of the microbiota within populations and individuals over time.These findings have implications for the design and interpretation of experiments involving laboratory animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
Genetics, diet, and other environmental exposures are thought to be major factors in the development and composition of the intestinal microbiota of animals. However, the relative contributions of these factors in adult animals, as well as variation with time in a variety of important settings, are still not fully understood. We studied a population of inbred, female mice fed the same diet and housed under the same conditions. We collected fecal samples from 46 individual mice over two weeks, sampling four of these mice for periods as long as 236 days for a total of 190 samples, and determined the phylogenetic composition of their microbial communities after analyzing 1,849,990 high-quality pyrosequencing reads of the 16S rRNA gene V3 region. Even under these controlled conditions, we found significant inter-individual variation in community composition, as well as variation within an individual over time, including increases in alpha diversity during the first 2 months of co-habitation. Some variation was explained by mouse membership in different cage and vendor shipment groups. The differences among individual mice from the same shipment group and cage were still significant. Overall, we found that 23% of the variation in intestinal microbiota composition was explained by changes within the fecal microbiota of a mouse over time, 12% was explained by persistent differences among individual mice, 14% by cage, and 18% by shipment group. Our findings suggest that the microbiota of controlled populations of inbred laboratory animals may not be as uniform as previously thought, that animal rearing and handling may account for some variation, and that as yet unidentified factors may explain additional components of variation in the composition of the microbiota within populations and individuals over time. These findings have implications for the design and interpretation of experiments involving laboratory animals.

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Comparison of variation within and among cages and shipment groups.(a) Principal coordinate plot of weighted UniFrac distances among microbiota from 46 mice sampled at 1–4 time points over a period of two weeks. Shapes of data points correspond to shipment group and colors of data points correspond to cage. Shipment p value was calculated using PERMANOVA on the factor of shipment group. Cage p value was calculated using PERMANOVA on the factor of cage. Cage(Shipment) p value was calculated using a PERMANOVA model where the factor of cage was nested within the factor of shipment group. (b) The average pairwise weighted UniFrac distance of samples from different mice within the same cage, between samples from mice in different cages, between samples from mice within the same shipment group, and between samples from mice in different shipment groups, across the entire population of mice. The average distance for each category was statistically significantly different from that of the technical replicates (includes all replicates—biological, extraction, sequencing run, barcode, and barcode/run). *** indicates p<0.001. ** indicates p<0.01. NS indicates p>0.05. (c) The average pairwise weighted UniFrac distance between samples from the same mouse, between samples from different mice, between samples from mice within the same cage, and between samples from mice in different cages within each shipment group. *** indicates p<0.001. NS indicates p>0.05.
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pone.0142825.g004: Comparison of variation within and among cages and shipment groups.(a) Principal coordinate plot of weighted UniFrac distances among microbiota from 46 mice sampled at 1–4 time points over a period of two weeks. Shapes of data points correspond to shipment group and colors of data points correspond to cage. Shipment p value was calculated using PERMANOVA on the factor of shipment group. Cage p value was calculated using PERMANOVA on the factor of cage. Cage(Shipment) p value was calculated using a PERMANOVA model where the factor of cage was nested within the factor of shipment group. (b) The average pairwise weighted UniFrac distance of samples from different mice within the same cage, between samples from mice in different cages, between samples from mice within the same shipment group, and between samples from mice in different shipment groups, across the entire population of mice. The average distance for each category was statistically significantly different from that of the technical replicates (includes all replicates—biological, extraction, sequencing run, barcode, and barcode/run). *** indicates p<0.001. ** indicates p<0.01. NS indicates p>0.05. (c) The average pairwise weighted UniFrac distance between samples from the same mouse, between samples from different mice, between samples from mice within the same cage, and between samples from mice in different cages within each shipment group. *** indicates p<0.001. NS indicates p>0.05.

Mentions: We hypothesized that mice received from the vendor in the same shipment would have more similar microbiota than mice received in different shipments. The mice used in this study were obtained from Jackson Laboratories, where mice of approximately the same age (3 weeks, +/- 3 days) from multiple litters were pool-weaned and maintained in the same weaning group prior to shipment (see Methods). While we do not know which, if any, of the mice were littermates (this information was not available), we do know that mice from the same shipment were housed together during weaning, the crucial period during which it is believed that the microbiota begins to develop a more adult-like profile [59,60]. Fecal specimens from mice of the same shipment tended to cluster on a PCoA plot where the two principal components (PC1 and PC2) together accounted for 70% of the variability in the data (especially shipments I and III, versus II and IV) (Fig 4A). Bray-Curtis distance plots showed similar patterns and biplots suggest the differences in shipments are explained by OTUs (S5 Fig). PERMANOVA indicated that microbiota composition differences among mice from different shipment groups were statistically significant (p = 0.001). Furthermore, the average pair-wise weighted UniFrac distance between specimens from mice of the same shipment was significantly lower than the average distance between specimens from mice of different shipments (Fig 4B). Strikingly, the range of microbiota compositions from shipments I and III were similar to each other, and likewise the ranges of microbiota compositions from shipments II and IV were similar to each other, even though each of these pairs of shipments was separated in time by several months.


Variation in Taxonomic Composition of the Fecal Microbiota in an Inbred Mouse Strain across Individuals and Time.

Hoy YE, Bik EM, Lawley TD, Holmes SP, Monack DM, Theriot JA, Relman DA - PLoS ONE (2015)

Comparison of variation within and among cages and shipment groups.(a) Principal coordinate plot of weighted UniFrac distances among microbiota from 46 mice sampled at 1–4 time points over a period of two weeks. Shapes of data points correspond to shipment group and colors of data points correspond to cage. Shipment p value was calculated using PERMANOVA on the factor of shipment group. Cage p value was calculated using PERMANOVA on the factor of cage. Cage(Shipment) p value was calculated using a PERMANOVA model where the factor of cage was nested within the factor of shipment group. (b) The average pairwise weighted UniFrac distance of samples from different mice within the same cage, between samples from mice in different cages, between samples from mice within the same shipment group, and between samples from mice in different shipment groups, across the entire population of mice. The average distance for each category was statistically significantly different from that of the technical replicates (includes all replicates—biological, extraction, sequencing run, barcode, and barcode/run). *** indicates p<0.001. ** indicates p<0.01. NS indicates p>0.05. (c) The average pairwise weighted UniFrac distance between samples from the same mouse, between samples from different mice, between samples from mice within the same cage, and between samples from mice in different cages within each shipment group. *** indicates p<0.001. NS indicates p>0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4643986&req=5

pone.0142825.g004: Comparison of variation within and among cages and shipment groups.(a) Principal coordinate plot of weighted UniFrac distances among microbiota from 46 mice sampled at 1–4 time points over a period of two weeks. Shapes of data points correspond to shipment group and colors of data points correspond to cage. Shipment p value was calculated using PERMANOVA on the factor of shipment group. Cage p value was calculated using PERMANOVA on the factor of cage. Cage(Shipment) p value was calculated using a PERMANOVA model where the factor of cage was nested within the factor of shipment group. (b) The average pairwise weighted UniFrac distance of samples from different mice within the same cage, between samples from mice in different cages, between samples from mice within the same shipment group, and between samples from mice in different shipment groups, across the entire population of mice. The average distance for each category was statistically significantly different from that of the technical replicates (includes all replicates—biological, extraction, sequencing run, barcode, and barcode/run). *** indicates p<0.001. ** indicates p<0.01. NS indicates p>0.05. (c) The average pairwise weighted UniFrac distance between samples from the same mouse, between samples from different mice, between samples from mice within the same cage, and between samples from mice in different cages within each shipment group. *** indicates p<0.001. NS indicates p>0.05.
Mentions: We hypothesized that mice received from the vendor in the same shipment would have more similar microbiota than mice received in different shipments. The mice used in this study were obtained from Jackson Laboratories, where mice of approximately the same age (3 weeks, +/- 3 days) from multiple litters were pool-weaned and maintained in the same weaning group prior to shipment (see Methods). While we do not know which, if any, of the mice were littermates (this information was not available), we do know that mice from the same shipment were housed together during weaning, the crucial period during which it is believed that the microbiota begins to develop a more adult-like profile [59,60]. Fecal specimens from mice of the same shipment tended to cluster on a PCoA plot where the two principal components (PC1 and PC2) together accounted for 70% of the variability in the data (especially shipments I and III, versus II and IV) (Fig 4A). Bray-Curtis distance plots showed similar patterns and biplots suggest the differences in shipments are explained by OTUs (S5 Fig). PERMANOVA indicated that microbiota composition differences among mice from different shipment groups were statistically significant (p = 0.001). Furthermore, the average pair-wise weighted UniFrac distance between specimens from mice of the same shipment was significantly lower than the average distance between specimens from mice of different shipments (Fig 4B). Strikingly, the range of microbiota compositions from shipments I and III were similar to each other, and likewise the ranges of microbiota compositions from shipments II and IV were similar to each other, even though each of these pairs of shipments was separated in time by several months.

Bottom Line: However, the relative contributions of these factors in adult animals, as well as variation with time in a variety of important settings, are still not fully understood.Our findings suggest that the microbiota of controlled populations of inbred laboratory animals may not be as uniform as previously thought, that animal rearing and handling may account for some variation, and that as yet unidentified factors may explain additional components of variation in the composition of the microbiota within populations and individuals over time.These findings have implications for the design and interpretation of experiments involving laboratory animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT
Genetics, diet, and other environmental exposures are thought to be major factors in the development and composition of the intestinal microbiota of animals. However, the relative contributions of these factors in adult animals, as well as variation with time in a variety of important settings, are still not fully understood. We studied a population of inbred, female mice fed the same diet and housed under the same conditions. We collected fecal samples from 46 individual mice over two weeks, sampling four of these mice for periods as long as 236 days for a total of 190 samples, and determined the phylogenetic composition of their microbial communities after analyzing 1,849,990 high-quality pyrosequencing reads of the 16S rRNA gene V3 region. Even under these controlled conditions, we found significant inter-individual variation in community composition, as well as variation within an individual over time, including increases in alpha diversity during the first 2 months of co-habitation. Some variation was explained by mouse membership in different cage and vendor shipment groups. The differences among individual mice from the same shipment group and cage were still significant. Overall, we found that 23% of the variation in intestinal microbiota composition was explained by changes within the fecal microbiota of a mouse over time, 12% was explained by persistent differences among individual mice, 14% by cage, and 18% by shipment group. Our findings suggest that the microbiota of controlled populations of inbred laboratory animals may not be as uniform as previously thought, that animal rearing and handling may account for some variation, and that as yet unidentified factors may explain additional components of variation in the composition of the microbiota within populations and individuals over time. These findings have implications for the design and interpretation of experiments involving laboratory animals.

Show MeSH
Related in: MedlinePlus