Metagenomic Characterization of Gut Microbiota in Children with Autism Spectrum Disorder: Microbial Signatures and Modulation by Anti-Inflammatory Diet and Probiotics

<b>Background:</b> Autism Spectrum Disorder (ASD) is increasingly associated with alterations in gut microbiota, intestinal permeability, and immune dysregulation. However, integrative studies exploring these mechanisms in Latin American populations are lacking. <b>Objective:</b> To characterize gut microbiota profiles in Colombian children with ASD and evaluate the effects of two microbiota-targeted interventions, an anti-inflammatory diet and a probiotic formulation, on microbial diversity and taxonomic composition. <b>Methods:</b> In a two-phase study, shotgun metagenomic sequencing was performed on fecal samples from 23 children with ASD and 7 typically developing (TD) controls. In the second phase, 17 children with ASD were randomized to receive a 12-week intervention (anti-inflammatory diet, probiotics, or no intervention). Alpha diversity indices (Shannon, Pielou, and Chao1) and differential abundance analyses were conducted. <b>Results:</b> Compared to TD children, those with ASD showed a higher <i>Firmicutes/Bacteroidetes</i> ratio and a significantly increased abundance of genera such as <i>Clostridioides</i>, <i>Thomasclavelia</i>, <i>Alistipes</i>, and <i>Coprococcus</i>. The presence of functional gastrointestinal disorders (FGIDs) in ASD patients is associated with reduced microbial richness. POST-intervention, the anti-inflammatory diet group showed that no statistically significant changes in alpha diversity were observed, although a slight upward trend was noted and significant enrichment of six bacterial genera, including <i>Moraxella</i> and <i>Eubacterium</i>. The probiotic group exhibited a significant increase in <i>Romboutsia</i> and a decrease in <i>Lachnospira</i>. Cytokine-microbiota networks in ASD were fragmented and dominated by IFN-γ and MCP-1 hubs, indicating systemic immune activation. Interventions induced functional remodeling: The anti-inflammatory diet increased the number of beneficial genera (<i>Eubacterium</i>, <i>Adlercreutzia</i>) and shifted networks toward positive correlations involving IL-8 and MIP-1β. Probiotics increased <i>Romboutsia</i>, reduced <i>Lachnospira</i>, and restructured networks with regulatory cytokines (SDF-1α, Eotaxin) and SCFA-producing taxa (<i>Blautia</i>, <i>Roseburia</i>). <b>Conclusions:</b> Children with ASD in Colombia displayed distinct microbial profiles characterized by pro-inflammatory taxa and altered richness. Both the anti-inflammatory diet and probiotics produced compositional shifts in the gut microbiota, although global changes in diversity were limited. These findings support the potential of microbiota-targeted nutritional strategies for ASD and underscore the need for precision interventions tailored to specific clinical and microbial phenotypes.