Article Figures & Data
Figures
- FIG 1
Biodiversity plot of sOTUs as relative frequencies at the taxonomic level of class. First-order shifts in microbial community composition, as revealed by 16S rRNA gene community profiling, from a predominance of Bacteroidetes to Tenericutes and return to Bacteroidetes was observed. Each column displays the relative bacterial community composition in a mouse fecal sample collected daily and sorted by the chronology of the experiment (i.e., day of experiment; Table 1). The columns are further sorted by group (i.e., group A, group B, and group C) and individual mice within each group (mouse 1, mouse 2, and mouse 3). Stacked bars are presented as relative frequencies at the taxonomical level of class, however, annotations of key taxa are at the phylum level (Bacteroidetes [green], Firmicutes [gray], and Tenericutes [fuschia]) or order level (Lactobacillales [yellow]).
- FIG 2
Diversity analyses. (A) Principal-coordinate analysis plot of unweighted UniFrac distances. Data points are projected onto the sample space and colored by pre-VREfm colonization (red), and post-VREfm colonization (blue). Note that circles and ellipses function to highlight the separation of experimental phases and do not indicate statistical confidence intervals. Principal coordinate axis 1 explains 41.66% of the variation observed between the naive microbiota and those from the post-VREfm colonization phase. (B) Community richness of the murine gut microbiome, as measured by Faith’s phylogenetic diversity, in response to ceftriaxone treatment and challenge with VREfm; (C) Community dissimilarity distances, as calculated by unweighted UniFrac, of each time point relative to day 0 (naive phase).
- FIG 3
Multinomial regression. Multinomial regression identified an Enterococcus exact sequence variant as the most positively associated with the colonization phase (log fold change score of 1.6693). Read counts for the Enterococcus ESV tracked daily across the experiment showing high abundance during the days of VREfm challenge.
- FIG 4
Metabolomic analyses. (A) Emperor plot displaying principal-coordinate analysis of binary Jaccard distances of metabolomic profiles. Samples are color coded, and the colors represent the naive (orange), antibiotic treatment (red), antibiotic weaning (blue), early VRE colonization (green), and late VREfm colonization (purple) phases. (B) Random Forest classifier identifying metabolite features (spectra) for each phase of the experiment. The heatmap is color coded from low ranking score (white; i.e., lowest importance) to high ranking score (dark blue; highest importance). Metabolite features are labeled by their mass-charge ratios and retention times for the reason that current databases do not capture their chemical structure and/or identifications. Abx tx, antibiotic treatment. (C) Peak quantification values for feature 6325 (m/z = 172.0671 and RT = 18.39) present in abundance during VRE colonization late (phase 4). (D) Peak quantification values for ceftriaxone (m/z = 555.0537 and RT = 13.30) tracked across the experiment. Ceftriaxone values are highest during antibiotic treatment (phase 2) and begins to wane during antibiotic weaning (phase 3).
- FIG 5
Microbe-metabolite vector biplots. Sequential biplots highlighting the changing metabolite differentials across each key phase of the experiment; Abx tx is the antibiotic treatment phase, and Abx wean is the period when antibiotics were removed for a 24-h period prior to colonization with VREfm. Each point on the sample space represents metabolites, and arrows represent microbes. Microbe and metabolite features are fixed upon the sample space, with gradient coloring of metabolites indicating the transition across key phases of the experiment. The distance between each point is indicative of metabolite cooccurrence frequency, and the angle between arrows indicates microbial cooccurrence. The directionality of the arrows describes the variation in the metabolites explained by the microbes represented by the arrows. For example, metabolite feature 6325 (m/z 173.067 and RT 18.392) is demonstrated to cooccur with Bacteroides. Information about the abundances of these cooccurring features are provided as heatmaps in Fig. S6 in the supplemental material.
Tables
- TABLE 1
Summary of samples analyzed in this study
↵a The key phases of the experiment where N represents naive, Abx-Tx represents antibiotic treatment, Abx-Wn represents antibiotic weaning, VRE-E represents early-phase post-VREfm colonization, and VRE-L represents late-phase post-VREfm colonization.
↵b Symbols: ✓, sample processed; −, data unavailable.
↵c The average number of sOTUs observed across all mice for each day of the experiment.
FIG S1
Alpha rarefaction plot of all samples rarefied to 20,000 reads per sample. Download FIG S1, PDF file, 3.5 MB.
Copyright © 2020 Mu et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
PERMANOVA testing (999 permutations) on unweighted UniFrac distances (16S rRNA gene data) relative to samples from the naive phase. P values for pairwise PERMANOVA testing are given in parentheses for the following phases of the experiment: naive and antibiotic treatment (0.001), naive and antibiotic treatment (0.001), naive and infection (0.001), and antibiotic treatment and antibiotic wean (0.025). Download FIG S2, PDF file, 0.3 MB.
Copyright © 2020 Mu et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S3
Emperor plot of procrustes analysis of unweighted (blue) and weighted (red) UniFrac distance matrices. Download FIG S3, PDF file, 1.3 MB.
Copyright © 2020 Mu et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S4
Pairwise PERMANOVA testing (999 permutations) on binary Jaccard distances (metabolome data) relative to samples from the naive phase. While naive and late VRE samples are significantly different, late VRE has a lower distance to naive compared to Abx txt, Abx wean, and early VRE samples. Download FIG S4, PDF file, 0.2 MB.
Copyright © 2020 Mu et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S5
Emperor visualization displaying samples assayed for metabolomics. Principal coordinate analysis plot of distances between each sample based on their overlapping molecules as measured by binary Jaccard. Samples assayed include pooled biological quality control samples (purple) and samples of known standard metabolite mixtures (green). The general metabolome profile of test samples is retained. Download FIG S5, PDF file, 0.7 MB.
Copyright © 2020 Mu et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S6
Paired feature abundance heatmaps. (A) Microbe abundances and (B) metabolite log centered abundances across each phase of the experiment. Heatmaps are aligned along the x axis (phase of experiment); the y axis displays microbe and metabolite abundances. Download FIG S6, PDF file, 0.4 MB.
Copyright © 2020 Mu et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
