Differences in means were tested by linear mixed effects with donor diagnosis as a fixed effect and donor ID, experimental stage, and test date as random effects. N oTD=10, N oASD=14 (2–4 male mice per donor). (A–B) Gastrointestinal physiology in offspring mice as measured by (A) Mouse weight at 12 weeks of age, (B) intestinal permeability as measured by FITC-Dextran (4 KDa) in serum following gavage. Gastrointestinal physiology and immunity are similar in offspring mice colonized with human ASD microbiomes, compared to TD microbiomes. Data presented are the aggregate of all experiments.ģ: Figure S3. α = 0.025 for Bonferroni-corrected statistical significance. N oASD= 121, N oTD= 85 (8–23 mice per donor, per sex). Hypothesis testing for differences of the means were done by a linear mixed effects analysis and p-values from a chi-square test. (F-H) Behavioral outcomes in offspring of colonized mice from selected eight donors stratified by mouse sex: (F) Repetitive behavior by marble burying test, (G) time socializing in direct social interaction, and (H) distance traveled in open field testing in colonized offspring colored by donor. Data presented is the aggregate of all experiments. Data stratified by mouse sex is presented in (A’, B’, C’, and D’) where α = 0.025 for Bonferroni-corrected statistical significance. N oASD= 179, N oASD-Mild= 44, N oTD= 111 (4–23 mice per unique donor per sex). Hypothesis testing for differences of the means were done by a random effects analysis and p-values from a chi-square test. ASD-Mild defined as StdADOS = 4–5, and ASD defined as StdADOS = 6–10 ( Gotham et al. (E) communication by ultrasonic vocalization (USV) in offspring of mice colonized with human samples from 16 donors, colored by donor. (C-D, C’-D’) distance traveled and center duration by open field testing. (A, A’) Repetitive behavior by marble burying test (A-E, A’-D’) Behavioral outcomes in offspring of colonized mice from 16 donors: Colonization with human ASD microbiomes reproduces behavioral deficits in mice. Differences in means were tested by linear mixed effects with donor diagnosis as a fixed effect and donor ID as a random effect.Ģ: Figure S2. (K) Aerobic and (L) anaerobic viable counts by plating on Tryptic Soy blood agar or Brucella blood agar, respectively. (J) Total 16S rRNA gene was measured by qPCR and quantified by a calibration curve with E. (J-L) Fecal bacterial load in offspring of colonized male mice. Differences in means were tested by linear mixed effects with donor diagnosis as a fixed effect and experimental stage, donor ID, and mouse sex as random effects. The fraction of taxa present in mice and respective donor, as well as the cumulative relative abundance of shared taxa in the donor are plotted. (H-I) Taxa engraftment in mice at the species level from 16S rRNA gene sequencing. (F-G) Taxonomic profile in donors (D), recipients (P), and offspring (F1) at the phylum level by diagnosis (F) and by donor (G) from 16S rRNA gene sequencing. Group differences by experimental stage (as in Panel C donors, recipients, and offspring) or by donor diagnosis were tested by pairwise PERMANOVA. (E) First two axes of a PCoA of unweighted UniFrac distances from 16S rRNA gene sequencing of human TD and ASD donor population. Group differences tested by Kruskal-Wallis test. (D) Box plots of α-diversity, as measured by the number of observed species from 16S rRNA gene sequencing. Differences between samples were tested by pairwise PERMANOVA test. (C) Box plots of pairwise distances of donor, recipients, and offspring mice to donor samples by unweighted UniFrac distances from 16S rRNA gene sequencing. Dark symbols denote samples that were further studied in depth. Group differences were tested by pairwise PERMANOVA. (B) First three axes of a principal coordinate analysis (PCoA) of unweighted UniFrac distances from TD (circles) and ASD (squares) donors from 16S rRNA gene sequencing. Sixteen samples used downstream are in dark-grey. Differences in means tested by Kruskal-Wallis. (A) α diversity as measured by observed amplicon sequence variants (ASVs) from 16S rRNA gene sequencing in TD and ASD individuals from which donor samples in this study were used. Engraftment fidelity for colonization of mice with human microbiomes.
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