Oral microbiome activity in children with autism spectrum disorder

Abstract

Autism spectrum disorder (ASD) is associated with several oropharyngeal abnormalities, including buccal sensory sensitivity, taste and texture aversions, speech apraxia, and salivary transcriptome alterations. Furthermore, the oropharynx represents the sole entry point to the gastrointestinal (GI) tract. GI disturbances and alterations in the GI microbiome are established features of ASD, and may impact behavior through the "microbial-gut-brain axis." Most studies of the ASD microbiome have used fecal samples. Here, we identified changes in the salivary microbiome of children aged 2-6 years across three developmental profiles: ASD (n = 180), nonautistic developmental delay (DD; n = 60), and typically developing (TD; n = 106) children. After RNA extraction and shotgun sequencing, actively transcribing taxa were quantified and tested for differences between groups and within ASD endophenotypes. A total of 12 taxa were altered between the developmental groups and 28 taxa were identified that distinguished ASD patients with and without GI disturbance, providing further evidence for the role of the gut-brain axis in ASD. Group classification accuracy was visualized with receiver operating characteristic curves and validated using a 50/50 hold-out procedure. Five microbial ratios distinguished ASD from TD participants (79.5% accuracy), three distinguished ASD from DD (76.5%), and three distinguished ASD children with/without GI disturbance (85.7%). Taxonomic pathways were assessed using the Kyoto Encyclopedia of Genes and Genomes microbial database and compared with one-way analysis of variance, revealing significant differences within energy metabolism and lysine degradation. Together, these results indicate that GI microbiome disruption in ASD extends to the oropharynx, and suggests oral microbiome profiling as a potential tool to evaluate ASD status. Autism Res 2018, 11: 1286-1299. © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Previous research suggests that the bacteria living in the human gut may influence autistic behavior. This study examined genetic activity of microbes living in the mouth of over 300 children. The microbes with differences in children with autism were involved in energy processing and showed potential for identifying autism status.

Keywords: autism spectrum disorder; developmental delay; gastrointestinal disturbance; microbiome; oropharynx; saliva.

Figure 1.

The core oral microbiome. The 10 oral taxa with the highest transcriptional activity across all participants (n = 346) are shown. Relative abundance (x-axis) for all 10 taxa exceeded 0.5% of the oral microbiome, and each taxa was present in counts of 10 or more in at least 70% of samples (prevalence, shown in red-blue scale).

Figure 2.

The core oral phyla. Abundance of oral transcripts at the phylum level across all participants (n = 346) are shown as percentage of the total (A). Firmicutes (58%) was the most abundant phylum, followed by Proteobacteria (16%) and Bacteroides (11%). Among the Firmicutes phylum (B) Lactobacillales was most abundant (72.4%) order, followed by Bacillales (24.5%).

Figure 3.

Bray–Curtis beta diversity. Microbial diversity between participants was calculated using a homogeneity of group dispersions technique for autism spectrum disorder (ASD) (red; n = 180), typically developing (TD) (green; n = 106), and developmental delay (DD) (blue; n = 60) groups. There was a significant difference (P = 0.04, F = 3.25) between groups, with the greatest between-samples diversity in the TD group. This two-dimensional plot accounts for 38.3% of the variance among participants. Confidence intervals of 95% are shown by the colored ovals.

Figure 4.

Oral phyla abundance across autism spectrum disorder (ASD), typically developing (TD), and developmental delay (DD) children. The relative abundance of 16 oral phyla is shown for children with autism spectrum disorder (ASD; n = 180), typically developing (TD; n = 106), and nonautistic DD (n = 60). Nonparametric Kruskal–Wallis testing revealed significant differences false discovery rate (FDR < 0.05) among the three groups for Planctomycetes (χ² = 31.0, FDR = 3.2E-06), Cyanobacteria (χ² = 14.8, FDR = 0.005), and Calditrichaeota (χ² = 9.6, FDR = 0.04).

Figure 5.

Oral taxonomic profiles distinguish autism spectrum disorder (ASD) children from typically developing (TD) and developmental delay (DD) peers. A PLS-DA was used to visualize differences in taxonomic profiles at the species level between ASD, TD, and DD groups in two dimensions (A). A model accounting for 4% of the variance between groups resulted in partial separation of ASD participants (red) from TD (blue) and DD (green) peers. The 20 taxa most critical for group projection are shown, based on variable importance in projection score (B). The majority of these taxa (14) are reduced (green boxes) in ASD samples relative to TD and DD groups. Three taxa are elevated in ASD participants (red boxes) and three demonstrated intermediate expression patterns (yellow boxes).

Figure 6.

Transcriptional activity of oral taxa differentiates autism spectrum disorder (ASD) participants. The ability of taxonomic RNA profiles to identify ASD status was explored with multivariate logistic regression analyses and visualized on receiver operator characteristic curve. The first 50% of subjects in each comparison were used to build cross-validation (CV) curves (blue), that were tested in the remaining 50% of naïve holdout samples (pink). Five ratios, involving eight taxa differentiated ASD and typically developing (TD) children with an area under the curve (AUC) of 0.795 (95% Confidence interval (CI): 0.711–0.872) on CV and 0.796 on holdout testing (A). Three ratios, involving five taxa differentiated ASD and developmental delay (DD) children with an AUC of 0.770 (95% CI: 0.643–0.867) on CV and 0.765 on holdout testing (B). Finally, three ratios, involving five taxa identified ASD children with gastrointestinal (GI) disturbance relative to ASD peers without GI disturbance in both CV (AUC = 0.839; 95% CI: 0.759–0.958) and holdout models (AUC = 0.857) (C).