Biology subjects

Gustafson, K. L.

Publications and source records attributed to Gustafson, K. L..

4 recordsLinked to original sources

bioRxiv2024

Biological and technical variability in mouse microbiome analysis and implications for sample size determination

BackgroundThe gut microbiome (GM) affects host growth and development, behavior, and disease susceptibility. Biomedical research investigating the mechanisms by which the GM influences host phenotypes often involves collecting single fecal samples from laboratory mice. Many environmental factors can affect the composition of the GM in mice and while efforts are made to minimize these sources of variation, biological variation at the cage or individual mouse level and technical variation from 16S rRNA library preparation exist and may influence microbiome outcomes. Here we employed a hierarchical fecal sampling strategy to 1) quantify the effect size of biological and technical variation and 2) provide practical guidance for the development of microbiome studies involving laboratory mice. ResultsWe found that while biological and technical sources of variation contribute significant variability to microbiome alpha and beta diversity outcomes but their effect size is 3- to 30-times lower than that of the experimental variable in the context of an experimental group with high intergroup variability. After quantifying variability of alpha diversity metrics at the technical and biological levels, we then simulated whether sequencing multiple fecal samples from individual mice could improve effect size in a two-group experimental design. Collecting five fecal samples per mouse increased effect size achieving the maximum 5% reduction in the required number of animals per group. While reducing the number of animals required, sequencing costs were dramatically increased. ConclusionsOur data suggest that the effect size of biological and technical factors may contribute appreciable variability to an experimental paradigm with relatively low mean differences. Additionally, repeated sampling improves statistical power however, its application is likely impractical given the increased sequencing costs.

microbiology↗

bioRxiv2024

Fetal programming by the maternal microbiome of offspring behavior, and DNA methylation and gene expression within the hippocampus

BackgroundThe microorganisms colonizing the gastrointestinal tract of animals, collectively referred to as the gut microbiome, affect numerous host behaviors dependent on the central nervous system (CNS). Studies comparing germ-free mice to normally colonized mice have demonstrated influences of the microbiome on anxiety-related behaviors, voluntary activity, and gene expression in the CNS. Additionally, there is epidemiologic evidence supporting an intergenerational influence of the maternal microbiome on neurodevelopment of offspring and behavior later in life. There is limited experimental evidence however directly linking the maternal microbiome to long-term neurodevelopmental outcomes, or knowledge regarding mechanisms responsible for such effects. ResultsHere we show that that the maternal microbiome has a dominant influence on several offspring phenotypes including anxiety-related behavior, voluntary activity, and body weight. Adverse outcomes in offspring were associated with features of the maternal microbiome including bile salt hydrolase activity gene expression (bsh), abundance of certain bile acids, and hepatic expression of Slc10a1. In cross-foster experiments, offspring resembled their birth dam phenotypically, despite faithful colonization in the postnatal period with the surrogate dam microbiome. Genome-wide methylation analysis of hippocampal DNA identified microbiome- associated differences in methylation of 196 loci in total, 176 of which show conserved profiles between mother and offspring. Further, single-cell transcriptional analysis revealed accompanying differences in expression of several differentially methylated genes within certain hippocampal cell clusters, and vascular expression of genes associated with bile acid transport. Inferred cell-to-cell communication in the hippocampus based on coordinated ligand-receptor expression revealed differences in expression of neuropeptides associated with satiety. ConclusionsCollectively, these data provide proof-of-principle that the maternal gut microbiome has a dominant influence on the neurodevelopment underlying certain offspring behaviors and activities, and selectively affects genome methylation and gene expression in the offspring CNS in conjunction with that neurodevelopment.

microbiology↗

bioRxiv2023

Effect size of delayed freezing, diurnal variation, and hindgut location on the mouse fecal microbiome relative to a standardized biological variable

BackgroundWhile murine fecal collection is central to microbiome research, there are a number of practical considerations that may vary during fecal sample collection, including time to sample storage, time of day the sample is collected, and position within the colon during terminal collections. While the need to control these factors is recognized, the relative effect on microbial community of duration at room temperature, time of day, and hindgut position, in the context of a known biological variable, is unclear. To answer these questions, and assess reproducibility of results across different microbiome compositions, parallel experiments were performed to investigate the effect of those factors on the microbiome of age- and sex-matched isogenic mice colonized with two different vendor-origin microbiomes. Results16S rRNA amplicon sequencing data from flash-frozen fecal samples showed no statistical difference in alpha or beta diversity compared to samples incubated for 1, 2, 3, 4, 6, and 9 hours at room temperature. Overall, samples collected in the AM period showed greater richness and alpha-diversity compared to samples collected in the PM period. While a significant effect of time was detected in all hindgut regions, the effect increased from cecum to distal colon. When using two vendor-origin microbiomes as a biological variable, its effect size vastly outweighed the effect size of the time samples spent at room temperature, the time of day samples were collected, and the position within the colon from which samples were collected. ConclusionsThis study has highlighted multiple scenarios encountered in microbiome research that may affect outcome measures of microbial diversity and composition. Unexpectedly, delayed time to sample cold storage up to nine hours did not affect the alpha or global beta diversity of fecal sample. We then presented evidence of location- and time-dependent effects within the hindgut on microbial richness, diversity, and composition. We finally demonstrated a relatively low effect size of these technical factors when compared to a primary experimental factor with large intergroup variability.

microbiology↗

bioRxiv2022

Multi-omics analysis of mouse fecal microbiome reveals supplier-dependent functional differences and novel metagenome-assembled genomes

Host genetics, sex, and other within-source factors have been associated with characteristic effects on the fecal microbiome in mice, however, the commercial source of mice remains the dominant factor. Increasing evidence indicates that supplier-specific microbiomes in particular confer differences in disease susceptibility in models of inflammatory conditions, as well as baseline behavior and body morphology. However, current knowledge regarding the compositional differences between suppliers is based on 16S rRNA amplicon sequencing data, and functional differences between these communities remain poorly defined. Here, we applied a meta-omic (metagenomic and metatranscriptomic) approach to biomolecules (DNA/RNA) extracted from murine fecal samples representative of two large U.S. suppliers of research mice, which differ in composition, and influence baseline physiology and behavior as well as disease severity in mouse models of intestinal disease. We reconstructed high-quality metagenome-assembled genomes (MAGs), frequently containing genomic content unique to each supplier. These differences were observed both within pangenomes of dominant taxa as well as the epibiont Saccharimonadaceae. Additionally, transcriptional activity and pathway analyses revealed key functional differences between the metagenomes associated with each supplier, including differences in carbohydrate enzyme activity and dissimilatory sulfate reduction by sulfate-reducing bacteria (SRB). These data provide a detailed characterization of the baseline differences in the fecal metagenome of laboratory mice from two U.S. commercial suppliers suggesting that these functional differences are influenced by differences in the initial inoculum of colony founders, as well as additional taxa gained during growth of the production colony.

microbiology↗