Reduced incidence of Prevotella and other fermenters in intestinal microflora of autistic children

Abstract

High proportions of autistic children suffer from gastrointestinal (GI) disorders, implying a link between autism and abnormalities in gut microbial functions. Increasing evidence from recent high-throughput sequencing analyses indicates that disturbances in composition and diversity of gut microbiome are associated with various disease conditions. However, microbiome-level studies on autism are limited and mostly focused on pathogenic bacteria. Therefore, here we aimed to define systemic changes in gut microbiome associated with autism and autism-related GI problems. We recruited 20 neurotypical and 20 autistic children accompanied by a survey of both autistic severity and GI symptoms. By pyrosequencing the V2/V3 regions in bacterial 16S rDNA from fecal DNA samples, we compared gut microbiomes of GI symptom-free neurotypical children with those of autistic children mostly presenting GI symptoms. Unexpectedly, the presence of autistic symptoms, rather than the severity of GI symptoms, was associated with less diverse gut microbiomes. Further, rigorous statistical tests with multiple testing corrections showed significantly lower abundances of the genera Prevotella, Coprococcus, and unclassified Veillonellaceae in autistic samples. These are intriguingly versatile carbohydrate-degrading and/or fermenting bacteria, suggesting a potential influence of unusual diet patterns observed in autistic children. However, multivariate analyses showed that autism-related changes in both overall diversity and individual genus abundances were correlated with the presence of autistic symptoms but not with their diet patterns. Taken together, autism and accompanying GI symptoms were characterized by distinct and less diverse gut microbial compositions with lower levels of Prevotella, Coprococcus, and unclassified Veillonellaceae.

Figure 1. Comparison on bacterial richness and diversity between neurotypical and autistic children.

(A) Rarefaction curves showing unique OTUs at the 95% threshold (a box graph at the rarefied sequence number), comparison of (B) Chao1 estimators and (C) phylogenetic diversity (PD) index between neurotypical (blue-colored box) and autistic (red-colored box) groups at different similarity thresholds (*: P<0.05, **: P<0.01 by one-tailed Mann-Whitney test).

Figure 2. Distribution of 39 subjects based on relative abundance.

(A) The top 10 most abundant genera, (B) 4 most differentially abundant genera (Red-colored box: autistic children, blue-colored box: neurotypical children), (C) the genus Prevotella obtained by qPCR analysis (Red-colored box: autistic children, blue-colored box: neurotypical children), and (D) receiver operating characteristics (ROC) curve of the 4 genera that have the highest area under curve (AUC).

Figure 3. Comparison of gut microbiota within the genus Prevotella between neurotypical and autistic children.

(A) Heat map profile and dendrogram (A01-A19: autistic children, N01–N20: neurotypical children). A red, orange, and blue scale bar represents scores of autistic symptoms, GI problems, and a log scale of the percentile abundance from a total bacteria, respectively. (B) Phylogenetic tree within the genus Prevotella. (C) The weighted UniFrac analysis with Prevotella copri-like 16 OTUs. Jackknife counts over 50 out of 100 are shown.

Figure 4. Genus level comparison of gut microbiota between neurotypical and autistic children.

(A) Heat map profile and dendrogram of all identified genera (A01-A19: autistic children, N01–N20: neurotypical children). A red, orange, and blue scale bar represents scores of autistic symptoms, GI problems, and a log scale of the percentile abundance from a total bacteria, respectively. (B) Principal Component Analysis at the genus level from the autistic and neurotypical children. Blue- and red-, and black-colored dots represent neurotypical, autistic samples, and 16 selected genera, respectively. Three genera representing enterotypes (23) were identified in bold. (C) The gradient of Prevotella and Bacteroides through neurotypical and autistic children (*: P<0.05 by Mann-Whitney test).