Gut microbial culturomics identifies autism-associated <i>Shigella</i> and reveals species-level remodeling during fecal microbiota transplantation

ABSTRACT Autism spectrum disorder (ASD) has been repeatedly linked to gut microbiota alterations, yet mechanistic insight remains limited by the scarcity of ASD-specific cultured isolates. Here, we performed large-scale gut microbial culturomics on fecal samples from 41 children with ASD and 12 typically developing (TD) controls, generating 1,724 isolates across six phyla. Longitudinal culturomics profiling was further conducted in 17 ASD children undergoing a 9-week fecal microbiota transplantation (FMT) intervention. All isolates underwent 16S rRNA sequencing and non-redundant clustering to assess species-level diversity, ASD-TD differences, and microbial dynamics associated with clinical response. ASD children harbored a distinct culturable microbiota enriched for Shigella flexneri and Shigella boydii , whereas TD children were enriched in beneficial taxa, such as Bifidobacterium catenulatum subsp. and other health-associated species. Notably, 20 species isolated from ASD children and 20 from TD children were absent from major existing gut microbiota biobanks, thereby expanding the cultivable repertoire. Among FMT participants, clinical responders exhibited increased alpha diversity, progressive enrichment of TD-associated beneficial taxa, such as Bacteroides fragilis , Anaerostipes hadrus , Parabacteroides merdae , and Turicibacter sanguinis , and a marked reduction of ASD-associated Shigella flexneri and Shigella boydii , whereas non-responders showed minimal shifts. Acquisition of TD-enriched strains at week 9 was strongly correlated with clinical improvement, suggesting that species-level ecosystem remodeling may contribute to FMT efficacy. This work establishes one of the first ASD-focused gut microbial culturomics resources, identifies Shigella as a potential ASD-associated taxon, and provides foundational evidence and testable mechanistic hypotheses for future microbiome-based interventions in ASD. IMPORTANCE Most autism spectrum disorder (ASD) microbiome studies rely on sequencing, which identifies associations but lacks live strains needed for mechanistic tests. We cultured 1,724 isolates from ASD and typically developing (TD) children, providing an ASD-focused, strain-level resource. ASD samples showed a significantly higher prevalence of Shigella flexneri . Longitudinal profiling during fecal microbiota transplantation (FMT) showed that clinical responders gained TD-enriched taxa and lost Shigella spp., and these shifts correlated with symptom improvement. This resource enables functional assays and gnotobiotic studies with ASD-relevant strains and provides a foundation for rational microbiome-based interventions.