A case-control analysis: changes in gut microbiota composition in children with autism spectrum disorder

Gut microbiota dysbiosis has been increasingly implicated in autism spectrum disorder (ASD), with the gut-brain axis proposed as a potential mechanistic link. However, ASD-specific microbial signatures remain inconsistent across studies, and whether reported associations reflect primary dysbiosis or ASD-related confounders such as dietary restriction and gastrointestinal comorbidities remains debated.

A case-control study was conducted enrolling 54 children with ASD and 46 age- and sex-matched typically developing (TD) children. Full-length 16S rRNA gene sequencing was performed on the PacBio Sequel IIe third-generation platform using universal primers 27F/1492R, generating high-fidelity (HiFi) reads via on-instrument circular consensus sequencing (CCS). Gut microbiota differences were assessed using α- and β-diversity analyses, phylum-level composition, Gut Microbiome Health Index (GMHI), Microbial Dysbiosis Index (MDI), differential abundance testing, and LEfSe analysis.

No significant differences were observed in α-diversity between groups, whereas significant differences were identified in β-diversity, GMHI, and MDI. Phylum-level analysis revealed a significantly reduced Bacillota/Bacteroidota ratio in the ASD group. More than 80% of ASD samples had negative GMHI values vs. more than 75% of TD samples with positive values, demonstrating superior discriminative performance compared with traditional diversity indices. Differential abundance analysis identified 15 differentially abundant species: 10 enriched in the TD group, predominantly butyrate-producing bacteria and Bifidobacterium spp., and 5 enriched in the ASD group, predominantly Bacteroides-affiliated taxa. LEfSe confirmed 16 differentially abundant taxa (LDA score ≥ 3.0).

Children with ASD exhibited significant differences in gut microbiota composition compared with TD children, characterized by a functional compositional imbalance-systematic depletion of butyrate-producing bacteria and Bifidobacterium spp. alongside enrichment of specific Bacteroides members-rather than alterations in overall species diversity. These findings are consistent with gut-brain axis dysregulation in ASD, though the cross-sectional design precludes causal inference and the possibility that observed differences reflect ASD-related dietary behavior cannot be excluded. GMHI demonstrated superior discriminative performance compared with traditional diversity indices in this cohort, though its application in pediatric ASD populations remains exploratory and requires independent validation in larger multicenter cohorts.