A single early-in-life macrolide course has lasting effects on murine microbial network topology and immunity

Abstract

Broad-spectrum antibiotics are frequently prescribed to children. Early childhood represents a dynamic period for the intestinal microbial ecosystem, which is readily shaped by environmental cues; antibiotic-induced disruption of this sensitive community may have long-lasting host consequences. Here we demonstrate that a single pulsed macrolide antibiotic treatment (PAT) course early in life is sufficient to lead to durable alterations to the murine intestinal microbiota, ileal gene expression, specific intestinal T-cell populations, and secretory IgA expression. A PAT-perturbed microbial community is necessary for host effects and sufficient to transfer delayed secretory IgA expression. Additionally, early-life antibiotic exposure has lasting and transferable effects on microbial community network topology. Our results indicate that a single early-life macrolide course can alter the microbiota and modulate host immune phenotypes that persist long after exposure has ceased.High or multiple doses of macrolide antibiotics, when given early in life, can perturb the metabolic and immunological development of lab mice. Here, Ruiz et al. show that even a single macrolide course, given early in life, leads to long-lasting changes in the gut microbiota and immune system of mice.

Fig. 1

Effect of number and timing of antibiotic doses on intestinal microbial communities. a Study design: 5-day-old C57BL/6 pups were treated with one course of tylosin at P5 for 5 days (PAT1 group) through their mother’s milk, or with two additional doses at P27 and P36 for 3 days each (PAT3 group). Twelve-week-old dams were treated with one course at pup P5. Sample sizes for dams were n = 7 (PAT), n = 3 (control). Sample sizes for their pups were n = 12 (control), n = 17 (PAT1), n = 19 (PAT3). b Mean (±SEM) unrarified α-diversity using the phylogenetic diversity (PD) metric in fecal samples of dams and offspring (pups) over the course of the experiment. Solid lines represent female pups n = 3 (control), n = 8 (PAT1), n = 9 (PAT3) and dams; dotted lines are male pups; n = 9 (control), n = 9 (PAT1), n = 10 (PAT3). Statistical analysis performed using two-sample t-test with Monte Carlo permutations, for statistical significance, see Supplementary Table 1. c Unweighted UniFrac analysis of fecal specimens of pups and dams visualized by principal coordinate analysis (PCoA). The three components explain 42.3% of the total variance. d Intergroup unweighted UniFrac distances averaged over independently drawn sample pairs (subsampled without replacement and replicated 999 times) for the time points and groups shown in c, shading indicates initial period of antibiotic exposure (for statistical significance, see Supplementary Table 2). e Mean relative abundance of taxa in control, PAT1 and PAT3 groups over the course of the experiment in fecal (over time), cecal and ileal samples

Fig. 2

Administration of one or three macrolide courses alters ileal gene signatures and T-cell populations. a Heatmap with unsupervised clustering of FDR-corrected, differential ileal gene expression profiles using Nanostring technology (Control (n = 7), PAT1 (n = 8) and PAT3 (n = 9)). After one-way ANOVA, Tukey HSD multiple comparison testing and FDR-correction, 148 genes remained significantly different between the groups. b Mean (±SD) frequency of small intestine lamina propria lymphocytes after PAT1, PAT3 or control exposure. Cells were isolated at sacrifice from pups (mean P52) and their respective dams (P61). Populations were gated on live CD45⁺CD4⁺ cells, and representative frequencies of CD4⁺IL17A⁺ cells shown. c Frequency of splenic lymphocytes after PAT1, PAT3 or control exposure. Cells were isolated from pups at sacrifice, gated on CD4⁺ and CD8⁺ populations, and representative frequencies shown. Data compiled from three experiments (n = 9–19 mice/group). d Secretory IgA (μg/ml) (mean ± SD) in fecal samples of control and PAT-exposed dams at P27 and their pups at P27 and P40. e Serum IgA (μg/ml) (mean ± SD) in control and PAT-exposed mice in pup and dam samples at mean P52 or P61, respectively. For panels (b–e) significance testing performed using the Mann–Whitney non-parametric test or the Kruskal–Wallis non-parametric test used with Dunnett’s multiple comparisons test. f Volcano plots showing significant global ileal transcriptomic alterations in pups and dams, determined by RNAseq; n > 16,000 genes examined, control (n = 3) and PAT (n = 6) dams, respectively; control (n = 3) or PAT (n = 4) pups. g Differentially expressed genes in relation to PAT exposure in P52 pups, and their dams at P61, determined by RNAseq, and depicted by direction of differences. For all panels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Physiologic effects of PAT in germ-free and conventional specific pathogen-free mice. a Study design: Germ-free (GF) and Specific pathogen-free (SPF) C57BL/6 mice were treated with one course of tylosin on P5 for 5 days; SPF mice (control (n = 15), PAT (n = 13)), GF mice (control (n = 7)), (PAT (n = 6)). All mice were sacrificed at P50. b Mean (±SD) unrarified α-diversity using the phylogenetic diversity (PD) metric of fecal samples at P21 and cecal and luminal contents at P50 in control and PAT SPF mice. Statistical analysis performed using two-sample t-test with Monte Carlo permutations, for statistical significance, see Supplementary Table 1. c Unweighted UniFrac analysis of fecal specimens in male (darker circles) and female (lighter circles) PAT or control SPF mice, depicted by principal coordinate analysis (PCoA). The three components explain 58% in total variance. Statistical analysis performed using an Adonis test, p = 0.001. d Aggregate cecal size in SPF and GF mice at P50 (mean (±SD)). e Secretory IgA (mean (±SD)) in luminal contents of offspring at P50, SPF mice (control (n = 5), PAT (n = 6), GF mice (control (n = 3), PAT (n = 4)). f PCoA of differential gene expression profiles in GF and SPF Control and PAT-exposed groups, with 76.2 and 8.1 % of the variance represented in PC1 and PC2, respectively. g Heat map of unsupervised hierarchal clustering of differential ileal gene expression profiles. After one-way ANOVA, Tukey HSD multiple comparison testing and FDR-correction, 145 genes were differentially expressed either between control vs PAT SPF, or GF vs SPF control groups (n = 3/group). h Frequency of small intestine lamina propria lymphocyte (LPL) populations in PAT-treated GF and SPF mice (mean (±SD)). Intestinal LPLs were isolated from offspring at sacrifice. Representative flow cytometry plots were gated on live TCRβ⁺ cells, with frequencies of TCRβ⁺, IL17A⁺CD4⁺, and Rorγt⁺CD4⁺ cells shown. Data are compiled from two experiments. Statistical analysis performed using the Kruskal–Wallis non-parametric test with Dunnett’s multiple comparisons test. For all panels: *p < 0.05, **p < 0.01, ****p < 0.0001

Fig. 4

Dynamics of PAT-perturbed microbiota after transfer and effects on host immune phenotypes. a Study design: donor mice received non-acidified water alone (controls) or drinking water with tylosin (PAT) from P5 to P10 and were sacrificed at P12, then cecal contents were harvested and pooled. Germ-free (GF) C57BL/6 mice at P35 were gavaged with cecal contents from the P12 control or PAT-exposed donors (n = 7 per group). b Fecal secretory IgA from day 0 to 76 post-gavage in the now-conventionalized mice that had received control or PAT cecal contents. A random effects repeated measures analysis of variance (ANOVA) was used to test for differences in IgA values between the PAT and the control recipients, taking into account the correlated structure of the measurements within subjects, (F_1,10 = 43.36, p > 0.0001). c Frequency of splenic TCRβ⁺ lymphocytes at sacrifice, 77 days post-gavage, (mean (±SD)). Statistical analysis was performed using the Mann–Whitney U non-parametric test; *p < 0.05. d Relative abundance of taxa in inoculum (in), fecal, cecal (ce) and colon (co) samples over the course of the experiment. e Median (±IQR) unrarified microbiota α-diversity over the course of the experiment in recipients of the control or PAT-perturbed inoculum, in fecal samples, or ileal (il), cecal (ce) and colonic (co) contents at sacrifice. f Unweighted Unifrac analysis of inoculum and fecal specimens from groups conventionalized with control (blue) or PAT-perturbed (red) microbiota, visualized in principal coordinate analysis (PCoA); the three components explain 43% of the total variance for each panel. Statistical analysis of intergroup UniFrac distances performed by Adonis test, with p-values shown

Fig. 5

Network properties recapitulate experimental perturbation. Networks were inferred using the SPIEC-EASI pipeline. To compare graphs, a two-dimensional embedding of graphlet correlation distances (using multidimensional scaling (MDS)) was used with the network positions shown as circles (from the dosing experiment (see Fig. 1)) or triangles (from the transfer experiment (see Fig. 4)). The corresponding networks on the MDS plot were overlaid near their respective positions in the embedding, and shown in a force-directed layout. Within each graph, nodes are colored by OTU family, with size proportional to (geometric) mean abundance, and edge width proportional to confidence score, as determined by stability selection. Each panel shows the number of OTUs (identified as those present in > 20% of samples), corresponding number of edges and calculated edge density (predicted edges/number of pairs of nodes)

Fig. 6

Schematic of the experiments performed in this study. Footnotes: All + refer to samples collected from pups, except: a, samples collected from both pups and dams; b, samples for RNAseq also collected from both pups and dams; c, serum collected at sacrifice from both pups and dams; d, IgAseq also performed in both groups of pups