Table 2.

Evidence of microbiota alterations in ASD compared to NT siblings.

Study characteristics	Microbiota assessment	Increasing microbial taxa	Decreasing microbial taxa	Metabolites/Immune markers	Functional pathways
Son et al. (2015) Case–control (CASP Score: 14) Sample size: N = 103 Age range (years): 7-14 Dietary habits: Documented; included in the analysis GI symptoms: ↑pain and constipation in ASD-FGID symptoms in ASD and NT (4 groups)	16S rRNA sequencing (Stool samples) Alpha diversity No differences Beta diversity No differences	↑Cyanobacteria/Chloroplast in ASD-GI compared to ASD-No-GI, NT-GI, and NT-No-GI siblings (probably due to chia seeds intake in two ASD-GI with constipation)	No decrease was observed	Not investigated	Not investigated
Gondalia et al. (2012) Case–control (CASP Score: 13) Sample size: N = 104 Age range (years): 2-12 Dietary habits: Not investigated GI symptoms: ↑GI symptoms in ASD	GS FLX Titanium sequencing platform (Roche®, USA) (Stool samples) Alpha diversity No differences Beta diversity No differences	No increase was observed	No decrease was observed	Not investigated	Not investigated
De Angelis et al. (2013) Case–control (CASP Score: 20) Sample size: N = 30 Age range (years): 4-10 Dietary habits: Not investigated GI symptoms: Not investigated	16S rRNA sequencing (bTEFAP) and bacterial cultures (Stool samples) Alpha diversity ↑ number of observed species and evenness (Chao1 and Shannon indexes) in ASD and PDD-NOS Beta diversity Microbiota composition is different between ASD, PDD-NOS and NT	Phylum ↑Bacteroidetes in ASD ↑active Firmicutes in PDD-NOS Genus In ASD: ↑Clostridium Roseburia Caloramator Sarcina Akkermansia Shigella In PDD-NOS: ↑Ruminococcus Collinsella Faecalibacterium In ASD and PDD-NOS: ↑Anaerofilum Dorea Caloramator Bacteroides in ASD (and species B. fragilis, B. vulgatus) Alistipes	Phylum ↓total and active Firmicutes in ASD ↓Fusobacteria, Verrucomicrobia in ASD and PDD-NOS Genus In ASD: ↓Faecalibacterium (and species F. prausnitzii) Prevotella species Bifidobacterium (and species B. sp. and B. adolescentis) Collinsella In PDD-NOS: ↓Coprococcus Lachnospira in Prevotella (and both species P. sp. And P. copri) In ASD and PDD-NOS: ↓Oscillospira Sporobacter Subdoligranulum Escherichia Fusobacterium	Microbial-derived metabolites: Free amino acids (FAA): ↑total FAA in ASD compared to NT and PDD-NOS ↑Glu, Ala, Asp., Lys, Val, Ile, Phe, His, Trp, Lys and Pro in ASD and PDD-NOS compared to NT Volatile organic compounds (i.e., aldehydes, esters, sulfur compounds) in stool samples are different in ASD compared to PDD-NOS and NT SCFAs: ↓total medium and SCFAs in PDD-NOS and ASD ↑acetic and propionic acids in ASD and PDD-NOS Positive correlations: Clostridium species and methyl esters (of the butanoic acid, acetic acid and pentanoic acid) and indoles Faecalibacterium, Ruminococcus and Bifidobacterium genera with total SCFAs	Not investigated
Pulikkan et al. (2018) Case–control (CASP Score: 17) Sample size: N = 54 Age range (years): 3-16 Dietary habits: Documented; not included in the analysis GI symptoms: ↑GI symptoms in ASD	16S rRNA sequencing (Stool samples) Alpha diversity No differences Beta diversity No differences between ASD and NT Correlations of the covariates showed autism as a significant component in differentiating the gut microbiome of ASD and NT ↑Firmicutes/Bacteroidetes ratio in ASD	Family ↑Veillonelleaceae Lactobacillaceae Bifidobacteriaceae Erysipelotrichaceae Enterococcaceae Desulfovibrionaceae Genus ↑Bifidobacterium Lactobacillus (and L. ruminis) Megasphaera Mitsuokella Ruminococcus Coprococcus Butyrivibrio Klebsiella	Family ↓Prevotellaceae, Genus ↓Prevotella Faecalibacterium Roseburia	Possible outcomes: ↑Lactobacillus- > potential inhibition of IDO1 which can affect the Th17 cell functioning leading to inflammatory conditions ↓Faecalibacterium and Roseburia - > potential reduction in the production of SCFAs	Not investigated
Ahmed et al. (2020) Case–control (CASP Score: 8) Sample size: N = 131 (NT are both siblings and unrelated-2 groups) Age range (years): 2-12 Dietary habits: Documented; not included in the analysis GI symptoms: 80% of ASD with GI symptoms	16S rRNA sequencing (Stool samples) Alpha diversity No differences Beta diversity No differences ↓Firmicutes/Bacteroidetes ratio in ASD and the NT siblings Firmicutes/Bacteroidetes ratio is different between ASD and NT unrelated Firmicutes/Bacteroidetes ratio is not different between ASD and NT siblings	Phylum ↑Firmicutes in severe ASD compared to moderate and mild Genus ↑Bacteroides Ruminococcus Species ↑Bifidobacterium sp. in NT siblings compared to ASD and NT unrelated	Phylum No decrease was observed Genus ↓Prevotella ↓Prevotella to Bacteroides Ratio in ASD and NT siblings compared to NT unrelated Species No decrease was observed	Not investigated	Not investigated
Yap et al. (2021) Case–control (CASP Score: 18) Sample size: N = 247 (NT are both siblings and unrelated-2 groups) Age range (years): 2-17 Dietary habits: Documented and included in the microbiota analysis: Dietary diversity ↓dietary alpha diversity in ASD compared to NT siblings and NT unrelated -Positive correlation between microbial taxonomic and dietary diversity in all the groups ↓dietary diversity is associated with ↓microbiome diversity in ASD but not in NT siblings or NT unrelated GI symptoms: ↓stool consistency in ASD	Metagenomics sequencing (Stool sample) Bacteria Alpha diversity No differences Beta diversity No differences Virome: No differences	Species No increase was observed	Species ↓Romboutsia timonensis in ASD compared to NT siblings and NT unrelated	Not investigated	↓ microbial genes responsible for the metabolism of amino acids (L-glutamine, L-lysine, L-methionine, and L-threonine), purines and pyrimidines, carbohydrates (galactose), bacterial spore germination and dsDNA digestion in ASD compared to NT siblings and NT unrelated (MGPP against the MGENES) database

ASD, Autism Spectrum Disorder; ASD-GI, Autism Spectrum Disorder with GI symptoms; CASP, Critical Appraisal Skills Program; FGID, functional gastrointestinal disorders (according to ROME III criteria); GABA, Gamma-aminobutyric acid; GI, gastrointestinal; IDO1, indoleamine 2,3-dioxygenase; MGENES, Microba Genes database; MGPP, Microba Gene and Pathway Profiler; NT, neurotypical controls; NT-GI, neurotypical with GI symptoms; No-GI, no GI symptoms; PDD-NOS, Pervasive Developmental Disorder – Not Otherwise Specified; rRNA, ribosomal ribonucleic acid; SCFAs, short chain fatty acids; Th-17, T helper 17 cells; bTEFAP, Tag-encoded FLX amplicon pyrosequencing. *Ruminococcaceae renamed to Oscillospiraceae in 2019.