Shotgun Metagenomics Study Suggests Alteration in Sulfur Metabolism and Oxidative Stress in Children with Autism and Improvement after Microbiota Transfer Therapy

Abstract

Links between gut microbiota and autism spectrum disorder (ASD) have been explored in many studies using 16S rRNA gene amplicon and shotgun sequencing. Based on these links, microbiome therapies have been proposed to improve gastrointestinal (GI) and ASD symptoms in ASD individuals. Previously, our open-label microbiota transfer therapy (MTT) study provided insight into the changes in the gut microbial community of children with ASD after MTT and showed significant and long-term improvement in ASD and GI symptoms. Using samples from the same study, the objective of this work was to perform a deeper taxonomic and functional analysis applying shotgun metagenomic sequencing. Taxonomic analyses revealed that ASD Baseline had many bacteria at lower relative abundances, and their abundance increased after MTT. The relative abundance of fiber consuming and beneficial microbes including Prevotella (P. dentalis, P. enoeca, P. oris, P. meloninogenica), Bifidobacterium bifidum, and a sulfur reducer Desulfovibrio piger increased after MTT-10wks in children with ASD compared to Baseline (consistent at genus level with the previous 16S rRNA gene study). Metabolic pathway analysis at Baseline compared to typically developing (TD) children found an altered abundance of many functional genes but, after MTT, they became similar to TD or donors. Important functional genes that changed included: genes encoding enzymes involved in folate biosynthesis, sulfur metabolism and oxidative stress. These results show that MTT treatment not only changed the relative abundance of important genes involved in metabolic pathways, but also seemed to bring them to a similar level to the TD controls. However, at a two-year follow-up, the microbiota and microbial genes shifted into a new state, distinct from their levels at Baseline and distinct from the TD group. Our current findings suggest that microbes from MTT lead to initial improvement in the metabolic profile of children with ASD, and major additional changes at two years post-treatment. In the future, larger cohort studies, mechanistic in vitro experiments and metatranscriptomics studies are recommended to better understand the role of these specific microbes, functional gene expression, and metabolites relevant to ASD.

Keywords: autism spectrum disorder (ASD); fecal microbiota transplant (FMT); gut microbiome; metagenomics; microbiota transfer therapy (MTT).

Figure 1

Overview of MTT trial in ASD and study time points for this work. hd—high dose; ld—low dose; SHGM—standardized human gut microbiota; WGS—whole-genome sequencing; MB—metabolomics; CA—clinical assessment (includes all ASD- and GI-associated symptoms; movi—MoviPrep. For complete trial information, refer to [7,11,20].

Figure 2

Univariate comparison of the (z-score) relative abundance of gut bacteria for the ASD Baseline group vs. all other groups. Top 30 bacterial species in three clusters that had significantly different relative abundance at Baseline vs. TD (cutoff p < 0.01, adjusted p < 0.05). Median of bacterial relative abundance was used to construct the heatmap for each group. * Single asterisk indicates p < 0.05, ** double asterisks indicate p < 0.01, triple *** asterisks indicate p < 0.001. All p-values are FDR-corrected. ASD: autism spectrum disorder; TD: typically developing.

Figure 3

Univariate comparison of the relative abundance (after log₁₀ transformation) of (A) P. denatalis, (B) P. enoeca, (C) P. oris, and (D) P. meloninogenica of ASD Baseline vs. MTT (10 wk, 2 yr) and TD. Red-dashed lines represent the mean of maintenance (n = 2) and blue represent median of major donors (n = 5). Each colored dot represents one ASD individual and grey-colored dots represent TD. Asterisks represent significant differences between ASD Baseline and the other groups ** double asterisks indicate p < 0.01; ns: not significant; all p-values are FDR-corrected). ASD: autism spectrum disorder, TD: typically developing.

Figure 4

Univariate comparison of the relative abundance (after log₁₀ transformation) of different bacterial species: (A) Bifiodobacterium bifidum, (B) Desulfovibrio piger, (C) Lactobacillus vaginalis and (D) Alistipes finegoldii for ASD Baseline vs. all other groups. Red-dashed lines represent the mean of maintenance donors (n = 2) and blue represents the median of major donors (n = 5); for A. finegoldii, the red and blue lines overlap. Each colored dot represents one ASD individual and grey-colored dots represent TD. Asterisks represent significant differences between ASD Baseline and the other groups (* single asterisk indicates p < 0.05; ** double asterisks indicate p < 0.01; triple *** asterisks indicate p < 0.001; #: statistically significant only after log₁₀ transformation; ns: not significant; all p-values are FDR-corrected). ASD: autism spectrum disorder; TD: typically developing.

Figure 5

Heatmap of KOs that were significantly lower (KO Cluster-I) or higher (KO Cluster-II) in ASD baseline vs. TD. The heatmap also shows how KOs changed after MTT compared to Baseline, and generally became more similar to the TD group. The median of bacterial relative abundance was used to construct the heatmap for each group. * Single asterisk indicates p < 0.05, ** double asterisks indicate p < 0.01, triple *** asterisks indicate p < 0.001. Univariate statistical comparisons were made for ASD Baseline vs. all other groups. All p-values are FDR-corrected. ASD: autism spectrum disorder; TD: typically developing.

Figure 6

Univariate comparison of the relative abundance (after log₁₀ transformation) of gut microbiome genes/KOs that changed significantly after MTT in ASD and became more similar to gene profiles in TD. (A) K04094: folate-dependent ribothymidyl synthase, (B) K10254: oleate hydratase, (C) K02499: tetrapyrrole methylase. Red-dashed lines represent the mean of maintenance (n = 2) and blue represent the median of major donors (n = 5). Colored dots represent ASD individuals and grey-colored dots represent TD. Asterisks represent significant differences between ASD Baseline and the other groups (* single asterisk indicates p < 0.05; ** double asterisks indicate p < 0.01; triple *** asterisks indicate p < 0.001; ns: not significant; all p-values are FDR-corrected). ASD: autism spectrum disorder; TD: typically developing.

Figure 7

Univariate comparison of the relative abundance (after log₁₀ transformation) of gut microbiome genes/KOs that encode enzymes involved in oxygen detoxification and oxidative stress before and after MTT in ASD in comparison with TD. (A) Oxidative stress protection and detoxification of reactive oxygen species; K05919 (dfx gene, SOR): superoxide reductase. (B) K07304 (msrA): peptide-methionine (S)-S-oxide reductase. (C) Illustration of enzymatic reactions of SOR and msrA KOs. Dashed lines represent the median of donors. Red-dashed lines represent the mean of maintenance (n = 2) and blue represents the median of major donors (n = 5). Colored dots represent ASD individuals and grey-colored ones TD. Asterisks represent significant differences between ASD Baseline and the other groups (* single asterisk indicates p < 0.05; ** double asterisks indicate p < 0.01; ns: not significant; all p-values are FDR-corrected). ASD: autism spectrum disorder; TD: typically developing.

Figure 8

Univariate comparison of the relative abundance (after log₁₀ transformation) of genes encoding for enzymes involving microbial sulfur metabolism (dissimilatory sulfate reduction) before and after MTT in ASD in comparison with TD. (A) K01082 (BPNT1/cycQ) 3′(2), 5′-bisphosphate nucleotidase, (B) K00395 (aprB) adenylylsulfate reductase, subunit B. (C) Diagram Illustrating dissimilatory sulfur reduction and the contribution of BPNT1 and aprB to the process. Each colored dot represents an ASD individual and grey-colored dots represent TD. Red-dashed lines represent the mean of maintenance (n = 2) and blue represents the median of major donors (n = 5). Asterisks represent significant differences between ASD Baseline and the other groups (* single asterisk indicates p < 0.05; ** double asterisks indicate p < 0.01; ns: not significant; all p-values are FDR-corrected). ASD: autism spectrum disorder; TD: typically developing.

Figure 9

Overview illustration of microbiome and metabolic pathways/KOs in children with ASD before and after MTT. Gastrointestinal Symptom Rating Scale (GSRS); Childhood Autism Rating Scale (CARS).