Table 1.

ASD microbiota studies

Subject details	Methods	Findings	References
11 with regressive ASD (10M/1F); 3.5–7 years	Antibiotic Intervention Study Oral vancomycin 500 mg/day for 8 weeks, followed by 2 weeks daily probiotics (40 Billion CFU).	ASD behaviors improved after treatment with oral vancomycin. Gains were lost after treatment ended.	Sandler et al., 2000 [46]
13 with regressive ASD 8 TD; 3.5–7 years Gender not specified	Fecal, gastric/jejunum/duodenal juice samples: sPCR identification after culturing	Increased Clostridia in ASD. Spore formers found in upper GI of ASD, not present in TD	Finegold et al., 2002 [47]
15 ASD, 8 TD Age, gender not specified	Fecal samples: RT-qPCR with species or cluster specific TaqMan primers/probes	Increased Clostridia clusters I (9-fold increase) and IX (3.5-fold increase), increased C. bolteae (46-fold increase)	Song et al., 2004 [48]
58 ASD (48M/10F; 53 with GI) 12 SIB (7M/5F; 3 with GI) 10 TD (6M, 4F; no GI) 3–16 years	Fecal samples: fluorescence in situ hybridization (FISH) for bacterial identification	Increased Clostridia clusters I/II, increased C. histolyticum. High levels of Clostridia associated with worsening GI symptoms and probiotic use. Intermediate levels seen in siblings.	Parracho et al., 2005 [49]
33 ASD with GI (24M/9F) 7 SIB no GI (2M/5F) 8 TD no GI 5M/3F 2–13 years	Fecal samples: bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP)	Increased Bacteroidetes to Firmicutes ratio. Increased Desulfbvibibrio and Bacteroides vulgatas in severe ASD.	Finegold et al., 2010 [50]
15 ASD with GI 7 TD with GI All male 4–4.5 years	Ileal and cecal biopsies: RT-qPCR for expression of enzymes and transporters associated with carbohydrate digestion and transport. 16sRNA pyrosequencing of cecal and ileal tissues for bacterial communities	Reduced mRNA transcripts for disaccharidases and carbohydrate transporters, associated with decreased Bacteroidetes increased Firmicutes and beta-Proteobacteria	Williams et al., 2011 [51]
23 ASD (21M/2F) 22 SIB (11M/11F) 9 TD (4M/5F) 3–18.5 years	Fecal samples: qPCR with bacteria specific primers	Reduced Bifidobacteria and Akkermansia muciniphila in feces of ASD children.	Wang et al., 2011 [52]
58 ASD (50M/8F) 39 TD (18M/21F) 3–12 years	Fecal samples: culture technique for bacterial measurement. Stool chemistry testing via ELISA. SCFA measured via gas chromatography.	GI scores strongly correlated with severity of ASD Reduced Bifidobacteria, increased Bacillus and Lactobacillus. Reduced levels of SCFA including propionate, especially in those taking a daily probiotic. Reduced lysozyme	Adams et al., 2011 [53]
41 ASD (32M/9F) 10 TD (5M/5F) 3–18 years	Fecal samples: culture technique for Clostridia measurement. ELISA for enterotoxin and lactoferrin analysis	No enterotoxins found. Elevated FLA in ASD boys only, with GI issues occurring 3–4 times per year. Clostridia found in both ASD and TD, the majority in ASD were C. perfringens. Strains varied from previous Clostridia studies, may be a factor of geographic location (Poland).	Martirosian et al., 2011 [54]
51 ASD, 28 with GI (42M/9F) 53 TD siblings (19M/34F) 2–12 years	Fecal samples: bTEFAP pyrosequencing of 16S rDNA	No significant differences at phylum and genus levels for ASD compared to TD siblings. Comparisons also done based on severity of ASD and GI symptoms, no differences detected.	Gondalia et al., 2012 [55]
23 ASD with GI 9 TD with GI All male 4–4.5 years	Ileal and cecal biopsies - 16 s rRNA pyrosequencing and Sutterella-specific PCR, qPCR. Immunoblot for IgM/IgG reactivity	12/23 ASD positive for Sutterella, mainly S. wadsworthensis or S. stercoricanis. Immune response detected – IgG/IgM reactivity to S. wadsworthensis in 11 ASD children seen via Western blot. No positive controls.	Williams et al., 2012 [56]
23 ASD (21M/2F) 22 SIB (11M/11F) 9 TD (4M/5F) 3–18.5 years	Fecal samples: qPCR with bacteria specific primers	Elevated Sutterella in ASD, elevated Ruminococcus torque in ASD with GI, and associated with reduced Akkermansia muciniphila found previously.	Wang et al., 2013 [80]
10 ASD 10 PDD-NOS 10 TD 14M/16F; Dx not specified by sex 4–10 years	Fecal samples: bTEFAP pyrosequencing of 16S rDNA and rRNA. Metabolome measured by GC-MS/SPME.	Significant differences at the phylum levels among ASD vs. PDD-NOS vs. TD, highest diversity in ASD children. Faelcalibacterium and Rummococcus highest in PDD-NOS and TD; ASD high in Caloramator, Sarcina and Clostridium in ASD. Bacteroidetes higher in ASD and PPD-NOS, Sutterellaceaea and Enterobacteriaceae highest in ASD. Alistepes and Akkermansia also higher in PDD-NOS and ASD. Differences in metabolites in ASD vs. TD (refer to Table 2 for details).	DeAngelis et al., 2013 [64]
20 ASD with GI(18M/2F) 20 TD no GI (17M/3F) ASD 6.7 (±2.7) years TD 8.3 (±4.4) years	Fecal samples: bTEFAP pyrosequencing of 16S rDNA	Decreased overall diversity and richness in ASD, correlating with ASD severity. Reduced carbohydrate fermenters Prevotella, Coprococcus and unclassified Veillonellaceae.	Kang et al., 2013 [74]
59 ASD, 25 with GI (52M/7F) 44 SIB, 13 with GI (21M/23F) 7–14 years	Fecal samples: qPCR with bacteria specific primers, 16S rRNA gene sequencing	No composition or diversity differences between ASD and NT siblings. Several low abundance taxa at genus level associated with ASD and/or ASD with GI.	Son et al., 2015 [75]
10 ASD (9M/1F) SIB (7M/2F) TD (all male) 2–17 years	Probiotic intervention study: Oral probiotics give 3×/day for 4 months. Fecal samples: RT-PCR with primers specific for main phyla and several species. No behavior measurement post-treatment.	Higher GI scores in ASD and siblings, ASD severity associated with GI symptoms. Reduced ratio of Bacteroidetes to Firmicutes in ASD, increased Lactobacillus. Increased Desulfovibrio associated with severity of ASD. Daily probiotic supplementation increased Bacteroidetes to Firmicutes ratio and reduced Desulfovibrio and Bifidobacterium in ASD, and Clostridia trended lower.	Tomova et al., 2015 [76]
21 ASD with GI (19M/2F; 14.4 ± 1.1 years) 19 TD with GI (10M/9F; 16.1 ± 1.3 years)	Duodenal biopsies: 16S rRNA gene sequencing. Disaccharidase activity measured via Dahlqvist method	No differences in disaccharidase activity. No differences in diversity/species richness and evenness. Increased Burkholderia and decreased Neisseria, Bacteroides species and Escherichia coli in ASD. Clostridium species correlated with disaccharidase activity in ASD.	Kushak et al., 2017 [77]
14 ASD with GI (all male) 15 TD with GI (12M/3F) 6 TD no GI (all male) 3–18 years	Rectal biopsies: Biopsy supernatant for 16S rRNA gene sequencing for microbiota characterization, 5-HT metabolites measured via HPLC. Blood and biopsy supernatant for cytokines via Luminex.	ASD with GI had increased Clostridiales, decreased Dorea, Blautia, and Sutterella. Abdominal pain correlated significantly with IL-6, IL-1, IL-17A, and IFN-y. IL-6 and tryptophan highest in ASD with GI pain, serotonin metabolites were increased in all with GI. Inflammatory cytokines, tryptophan and serotonin correlated with Clostridiales in ASD.	Luna et al., 2017 [83]
40 ASD (31M/9F) 40 TD (28M/12F) 3.5–17 years	Fecal samples: Calprotectin levels measured by ELISA.  454 pyrosequencing	No differences in Calprotectin levels in feces. Reduced ratio of Bacteroidetes to Firmicutes and decreased Alistipes, Bilophila, Dialistei; Parabacteroides, and Veillonella, increased Collinsella, Corynebacterium, Dorea, and Lactobacillus in ASD. Greater than twice the abundance of Candida in ASD. High levels of Escherichia, Shigella and Clostridium cluster XVIII associated with constipation in ASD.	Strati et al., 2017 [78]
18 ASD (16M/2F) 20 TD (18M/2F) 7–16 years	FMT Intervention Study 2-week tx with oral vancomycin followed by bowel cleanse and extended FMT: high initial dose, then daily maintenance doses for 7–8 weeks. Periodic fecal samples and swabs: 16 s rRNA gene sequencing. Pre-, during and post behavioral assessments: PGI-III, CARS, ABC, SRS and VABS-II	Fecal microbiota transplant improved behavior and GI symptoms in ASD, increased levels of Bifidobacteria, Prevotella and Desulfovibrio TD controls were monitored, but not treated.	Kang et al., 2017 [79•]
21 ASD with GI (25M/4F) 29 ASD no GI (17M/12F) 7 TD with GI (6M/1F) 34 TD no GI (32M/2F) 3–12 years	Fecal and blood samples: PBMC stimulation and cytokine analysis of supernatant via Luminex. Fecal 16S rRNA gene sequencing.	Elevated inflammatory immune responses in ASD with GI. Balance of inflammatory vs regulatory cytokines skewed. ASD microbiota clustered differently than TD samples, increased Bacteriodaceae, Lachnospiraceae, Prevotellaceae and Ruminococcaceae in ASD with GI vs TD with GI. Differences seen in functional KEGG pathways in ASD compared to TD. Altered zonulin levels in ASD with GI, may suggest increased intestinal permeability.	Rose et al., 2018 [57]
23 ASD (22M/1F) 21 TD (15M/6F) 4–17 years	Fecal samples: 16S rRNA gene sequencing	Reduced diversity, reduced Prevotella copri, Feacalibacterium prausnitzii and Haemophilus parainfluenzae. Altered microbial metabolites (refer to Table 2 for details).	Kang et al., 2018 [73]

ASD autism spectrum disorder, regressive ASD child developed typically but lost skills and speech, usually between 15 and 30 months, TD unrelated typically developing control, SIB typically developing sibling, ASP Asperger’s disorder, GI (group) gastrointestinal symptoms, PDD-NOS pervasive development disorder-not otherwise specified, Dx diagnosis, GC gas chromatography, MS mass spectrometry, SPME solid-phase microextraction, FMT fecal microbiota transplant, Tx treatment, FLA fecal lactoferrin