The diagnosis of GI abnormalities is typically indicated by certain behavioral complications (Maenner et al., 2012). A previous study reported that GI abnormalities (assessed by the 6-GSI) significantly correlate with symptom severity in ASD (assessed by the autism treatment evaluation checklist) (Adams et al., 2011). Furthermore, constipation is the most common GI symptom observed in autistic children (Srikantha and Mohajeri, 2019). Moreover, the presence and intensity of abdominal pain have been directly associated with the severity of ASD core symptoms (Ding et al., 2017). Such findings suggested a gut–brain axis-mediated relationship between GI anomalies in ASD and behavioral output (Hsiao, 2014). In addition, GI abnormalities have shown a correlation with other ASD comorbidities, such as sleep difficulties, abnormal mood, and social deficits. In comparison with ASD patients without GI symptoms, it has been discovered that GI comorbidity in patients with ASD was associated with increased sleep issues, abnormal mood, argumentative, oppositional, defiant, or destructive behavior, anxiety, sensory responsiveness, rigid compulsive behaviors, self-injury, aggression, lack of expressive language, and social impairment (Nikolov et al., 2009).

Increasing evidence has shown ASD children with constipation have higher relative abundances of Escherichia/Shigella and Clostridium cluster XVIII (Strati et al., 2017), the order Fusobacteriales, the family Actinomycetaceae, and the genera Fusobacterium, Barnesiella, Coprobacter, Olsenella, and Allisonella (Liu et al., 2019), as well as lower Faecalibacterium prausnitzii, Bacteroides eggerthii, Bacteroides uniformis, Oscillospira plautii, and Clostridium (C.) clariflavum amount (Luna et al., 2017). Moreover, the lower abundance of Lactobacilli (Iovene et al., 2017) could be related to constipation in patients with ASD because its depletion was connected with chronic constipation in non-ASD children (Kushak et al., 2017). Patients with ASD who also had allergies had higher relative abundances of the phylum Proteobacteria in their stools, previously linked to autoimmune diseases (Kong et al., 2019). In addition, cecal Betaproteobacteria, ileal and cecal Firmicutes, and the Firmicutes/Bacteroidetes ratio appear to increase in association with food allergies (Williams et al., 2011). It was found that Firmicutes/Bacteroidetes ratio is negatively correlated with allergy/immune function in feces in ASD children (Kong et al., 2019). Turicibacter sanguinis, C. lituseburense, C. disporicum, C. aldenense, and O. plautii levels were higher in ASD children who experienced GI discomfort. Some bacteria may be associated with >1 GI symptoms, for instance, C. aldenense and O. plautii have been also identified in ASD patients with constipation (Luna et al., 2017). Interestingly, some ASD children have extremely high levels of certain bacteria that are positively connected with GI symptoms (i.e., Turicibacter sanguinis) (Kang et al., 2013). More recently, Parracho et al. (2005) demonstrated that ASD children have higher fecal content of the C. histolyticum group-known toxin producers (Hatheway, 1990) than healthy unrelated controls but not than healthy siblings. In addition, high levels of Clostridium species were substantially related to GI issues in patients with ASD, including those with and without GI symptoms.

The gut microbiota dysbiosis in autism usually results in immune system disorders (Doenyas, 2018). Interleukin-1 (IL-1), interleukin-6 (IL-6), interferon (INF), and tumor necrosis factor (TNF) are chemokines and cytokines that are released by the active immune system which may cross the blood–brain barrier. These mediators attach to brain endothelial cells, triggering immunological reactions (de Theije et al., 2011). A previous study found significantly higher IL-1, IL-6, and IL-8 plasma levels in the ASD group than in the typical development controls (Ashwood et al., 2011). In addition, the immune system is concentrated in and around the gut mucosa, where around 80% of it is located (Critchfield et al., 2011).

Patients with ASD have variable bacterial diversity. According to several studies, they have significantly decreased species diversity and richness (Carissimi et al., 2019; Ma et al., 2019), whereas other studies found the opposite (Finegold et al., 2010; De Angelis et al., 2013). The gut microbiota affects brain physiology through its differential metabolites (Figure 2). Patients with ASD have been shown to have an increase in the level of metabolites including SCFAs, p-cresol, and ammonia, in serum, urine, and fecal samples, which can cause behavioral symptoms and symptoms resembling autism by the vagal pathway (Forsythe et al., 2014). Among these, SCFAs, including acetic acid, propionic acid, butyrate, isobutyric acid, valeric acid, and isovaleric acid, have been considered the major signaling metabolites, which play a critical role in regulating catecholamine production throughout life and in preserving the neurotransmitter phenotype after birth, and have been shown to be important in ASD (Wang et al., 2012). However, some studies found lower levels of these SCFAs, except for propionic and acetic acid, in children with ASD. Clostridium and Bacteroidetes can produce propionic acid, which can penetrate the blood–brain barrier and cause autism-like behaviors, such as impaired and restricted social, behavior, and cognition, by modulating 5-Hydroxytryptamine (5-HT) and dopamine (DA) in the brain (Thomas et al., 2012). In addition, propionic acid decreases the levels of intracellular antioxidants such as GSH and superoxide dismutase and the production of pro-inflammatory cytokines (Wajner et al., 2004). Increased oxidative stress and inflammation are known to play an important role in the pathogenesis of ASD (Bjørklund et al., 2020). Children with autism have been shown to have higher levels of the microbial metabolite p-cresol and its conjugate p-cresyl sulfate in their urine samples. Clostridia species and Pseudomonas stutzeri strains may explain the high p-cresol levels (Altieri et al., 2011). In addition, increasing serum levels of 4-methylphenol, a minor aromatic metabolite generated by gut bacteria, causes ASD-like behavior and hippocampus impairment (Liu et al., 2022). Moreover, ASD patients’ urine contains higher levels of 3-(3-hydroxyphenyl)-3-hydroxypropionic acid, a phenylalanine metabolite generated by Clostridia spp., which may be responsible for the depletion of catecholamines that worsens stereotyped behavior and hyperactivity (Shaw, 2010). In addition, it has been connected to ASD-like behaviors in mouse models. Particularly, offspring of dams treated with the inflammatory molecule poly (I: C) show changes in gut microbiota composition and dysregulation of metabolite concentrations in the serum, including elevated levels of the microbial metabolite 4-ethylphenylsulfate, which led to anxiety-like behavior in mice otherwise untreated (Hsiao et al., 2013). In addition, 5-aminovaleric acid and taurine levels were reduced in recipient mice microbiota from persons with ASD, and both these metabolites can act as aminobutyric acid (GABA) receptor agonists (Sharon et al., 2019). In fact, in the BTBR T + Itpr3tf/J mouse model of ASD, treatment with these two metabolites was effective in reducing repetitive behaviors and improving sociability (Sharon et al., 2019). Tryptophan’s metabolite, indole, serves as a precursor for crucial chemicals including 5-HT and DA (De Angelis et al., 2013) and is able to be synthesized by Alistipes that are higher in individuals with anxiety and depression (Zhang et al., 2015), ultimately disrupting the serotonergic balance in the body. Therefore, an aberrant increase or decrease in gut microbiota-derived metabolites can worsen the symptoms of ASD.

There is evidence that probiotic supplementation improved the behavior of ASD children through the gut–brain axis (Table 1). The effect of probiotics on psychological conditions such as depression and anxiety is relatively well known (Ng et al., 2018). Children with autism who received vancomycin orally and probiotic Bifidobacterium supplements had significantly higher urine levels of 3-(3-hydroxyphenyl)-3-hydroxyproionic acid, 3-hydroxyphenylacetic acid, and 3-hydroxyhippuric acid (Xiong et al., 2016). The first metabolite can cause autistic symptoms by lowering catecholamine levels in the brain (Li and Zhou, 2016). Thus, the decreased levels of those metabolites may be responsible for improved eye contact and less constipation in children with autism (Xiong et al., 2016). A recent study found that probiotics could improve the brain activity of preschoolers with ASD. This was demonstrated by a reduction in frontopolar region power in the beta and gamma bands, a decrease in frontopolar region coherence in the same bands, and a change in frontal asymmetry using electroencephalography (EEG) (Billeci et al., 2022). Beta waves are connected to physiological activity, focus, analytical thought, and states of specific mental commitment or motor activities (Tallon-Baudry, 2003), whereas gamma waves are associated with working memory tasks and several early sensory reactions. When compared to typically developing persons, ASD brains’ resting EEGs frequently show enhanced beta and gamma spectral band activity (Nicotera et al., 2019). Abnormal GABAergic tone in the growth of plasticity and brain function is expected to be involved in the regulation of the EEG frequency bands, which may be partially responsible for the atypical increase in high-frequency bands in ASD (Baumgarten et al., 2016). One of the main features of the neurophysiology of ASD is an altered GABA (the CNS primary inhibitory neurotransmitter) pattern. Atypical brain excitation/inhibition balance, altered neuronal signaling, information processing, and responsive behavior, in particular, may be caused by the deficient inhibitory GABAergic signaling that characterizes patients with ASD (Foss-Feig et al., 2017). After probiotic supplementation, the brain activity of ASD children (showing an improvement in excitatory/inhibitory imbalance) suggested that probiotics can promote a change in brain activity in ASD children toward that of controls. Moreover, probiotic administration was found to promote a shift in brain connections toward a more typical pattern with respect to coherence and asymmetry. Importantly, probiotics could significantly improve the brain function of animals with ASD. For example, immunohistochemical analysis of brain tissues showed that B. longum CCFM1077 could ameliorate microglia activities in the cerebellum of autistic rats, as evidenced by the decreased IBA-1 protein expression (Kong et al., 2022). Furthermore, oral probiotics (containing B. bifidum, B. infantis, and L. helveticus) could inhibit MIA-induced decrease in PV⁺ neuron numbers in the PFC in adult offspring (Wang et al., 2019). In addition, treatment with Lactobacillus strains reversed the VPA-induced apoptosis and degeneration in the cerebellum (Sunand et al., 2020). All the aforementioned studies suggested that the recovery of brain function after probiotics treatment provides important evidence for the connection between the gut and the brain.

There is no clear explanation for the regulatory effects of probiotic supplementation on the gut–brain axis in humans, but there are numerous preclinical studies in animal models of ASD (Table 2). Probiotics have been shown to prevent Candida from colonizing the stomach (Romeo et al., 2011), and Bifidobacterium (B.) longum BB536 could modulate Clostridium (decreased the harmful C. perfringens and increased Clostridium cluster IV) populations and rescue social impairment in a rodent model of autism induced by PPA (Abuaish et al., 2021). Some Clostridium species generate p-cresol, which has been suggested as a potential urine biomarker for autism (Persico and Napolioni, 2013). Moreover, Lactobacillus (L.) plantarum ST-III could ameliorate the social deficits, self-grooming, and freezing times and increase the abundance of the beneficial Lachnospiraceae and decrease that of Alistipes in a mouse model of ASD (offspring of pregnant mice exposure to triclosan) (Guo et al., 2022). The gut microbiota contains several members of the Lachnospiraceae family, which has beneficial effects on human health (David et al., 2014), as they can increase the synthesis of the SCFAs acetate and butyrate (Byndloss et al., 2017) as well as boost the conversion of primary to secondary bile acids and reduce the generation of pro-inflammatory cytokines, being also crucial in supplying energy to the host (Smith et al., 2013). Tryptophan is transformed into indoles by Alistipes, which ultimately throws off the body’s serotonergic equilibrium. A previous study found a higher presence of Alistipes in depressed and anxious individuals (Zhang et al., 2015). Treatment with L. helveticus CCFM1076 significantly reduced Turicibacter abundance in the gut and increased butyric acid levels in the cecum contents of valproic acid (VPA)-treated rats (Kong et al., 2021). In the BTBR mouse model of autism, probiotic L. rhamnosus therapy favorably influences the microbiota–gut–brain axis favorably (Pochakom et al., 2022), as indicated by a reduction in behavioral deficits in social novelty preference, increased microbial richness, phylogenetic diversity, presence of potential anti-inflammatory (Anaeroplasma and Christensenellaceae) and butyrate-producing taxa (Acetatifactor, Lachnospiraceae, and Butyricicoccus), and elevation of 5-aminovaleric acid and choline in serum and in the prefrontal cortex (PFC), respectively. Moreover, a mixture of probiotics VSL#3 significantly improved sociability, social interaction, anxiety-liked behavior, and behavioral despair, while restoring the Bacteroidetes/Firmicutes ratio induced by prenatal VPA exposure (Adıgüzel et al., 2022).

Second, probiotics can modulate neuroactive compounds to attenuate ASD symptoms. Accumulating evidence has demonstrated that genetic and environmental risk factors converge to disturb the balance between glutamate (Glu)-mediated excitatory and γ-GABA-mediated inhibitory neurotransmission autism (Nelson and Valakh, 2015; Borisova, 2018). Probiotics can influence neurotransmitters such as γ-GABA, Glu, and 5-HT (Ng et al., 2018; Israelyan and Margolis, 2019). Tabouy et al. (2018) revealed that L. reuteri treatment decreased repetitive behaviors and increased GABA receptor gene expression (GABRA1, GABRA1, and GABRB1) and protein levels (GABRA1) in the hippocampus and the PFC of Shank3 mutant mice (a model of ASD). Moreover, treatment with Lactobacillus was shown to regulate emotional behavior and central GABA receptor expression via the vagus nerve (Bravo et al., 2011), which communicates connecting the brain and the gut, in a mouse. Probiotics that stimulate inhibitory neurotransmission (for example, by increasing GABA levels) may help restore the excitatory/inhibitory balance and recover the decreased social interaction associated with ASD (El-Ansary et al., 2018). In addition, daily L. helveticus CCFM1076 intake alleviates autistic-related features by regulating 5-HT anabolism and catabolism, balancing excitatory and inhibitory neurotransmitter release (as indicated by increased GABA in PFC and decreased Glu in serum, and PFC) in both the peripheral and CNS, and increasing oxytocin synthesis in the hypothalamus (Kong et al., 2021). 5-HT is produced in the gut and plays a central role in gut–brain connection (Owens and Nemeroff, 1994). Previously, 5-HT levels have been significantly correlated with GABA, Glu, and oxytocin, suggesting a vital role of 5-HT in the neuroendocrine network. Moreover, a single dose of oxytocin has been shown to regulate the 5-HT energy system, reduce anxiety (Neumann and Slattery, 2016), and help alleviate social dysfunction (Lawson et al., 2016). Another neuropsychiatric disease involves the altered neurotransmitter Glu (Shimmura et al., 2011). ACh is involved in learning and memory, attention, cognition, social interactions, and stereotypical behaviors (Avale et al., 2011; Karvat and Kimchi, 2014). In addition, L. reuteri treatment raised oxytocin levels in the brain, which improved behavioral aspects of brain function by stimulating the vagus nerve (Sgritta et al., 2019). Another study found that L. reuteri ingestion restored maternal high-fat diet-induced social deficits, oxytocin levels, and ventral tegmental area plasticity in offspring (Buffington et al., 2016). Furthermore, L. reuteri has been repeatedly shown to improve oxytocin-dependent behavior in several ASD mice models (Sgritta et al., 2019). Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor that promotes the development and survival of cholinergic, dopaminergic, and serotonergic neurons in their mature and growing stages (Croen et al., 2008). Working memory, hippocampal learning, and brain plasticity are all influenced by BDNF (Leung and Thuret, 2015). In addition, BDNF impacts GABA inhibitory interneurons, ultimately causing cognitive deficits (Maqsood and Stone, 2016). One previous study reported that daily Lactobacillus strains supplementation reversed autistic deficits and decreased BDNF levels in serum and acetylcholinesterase (AChE) and 5-HT in the brain of the VPA-induced prenatal model of autism (Sunand et al., 2020). Acetylcholine (Ach), hydrolyzed by AChE in the synaptic cleft (Croen et al., 2008), is involved in learning and memory, attention, cognition, social interactions, and stereotypical behaviors (Karvat and Kimchi, 2014). In a recent report, both the pure and mixed probiotics had beneficial effects against PPA-induced neurotoxicity shown by increased levels of alpha-melanocyte-stimulating hormone (α-MSH) levels, neurotensin, and β-endorphin in ASD of rodent model (Alghamdi et al., 2022). A remarkable decrease in α-MSH in different brain regions has been involved in the pathogenesis of social isolation (Theoharides and Doyle, 2008); in fact, re-socialization fully recovered α-MSH immunoreactivity attenuating anxiety-and depression-like behaviors (Tejeda et al., 2012). Neurotensin may act on the CNS as atypical neuroleptics (Petrie et al., 2005). β-endorphin, endogenous opioid peptides, may alter social behavior and result in autistic-like features. A probiotic mixture was shown to attenuate both the antibiotics and VPA-induced autistic behavioral symptoms (Mintál et al., 2022). In the BTBR mouse model of autism, probiotic L. rhamnosus administration decreased behavioral abnormalities in social novelty preference and increased 5-aminovaleric acid and choline levels in serum and the PFC, respectively (Pochakom et al., 2022). The excitatory/inhibitory imbalance previously linked to the pathophysiology of ASD is attenuated by 5-aminovaleric acid, a GABA receptor agonist, of which persons with ASD have remarkably lower levels than non-ASD ones (Sharon et al., 2019). The social and behavioral impairments observed in ASD have been connected to cholinergic pathways through choline metabolism (Lam et al., 2006). Choline supplementation during pregnancy and blocking Ach the breakdown both helped BTBR mice with social and repetitive/restricted behavior deficiencies (Eissa et al., 2020).

The reduction of gut inflammation (improved immune functions) may be another benefit of probiotic application for ASD. Several GI illnesses, including irritable bowel syndrome and inflammatory bowel disease, have been associated with increased mucosal inflammation (Ng et al., 2018). Children with ASD have been found to have greater levels of gut immune inflammation, which is linked to gut dysbiosis, as well as GI complaints (Hughes et al., 2018). In fact, 4 months of probiotic supplementation in children with ASD aged 2–9 years restored many of the abnormalities in their GI microbiota and reduced their intestinal inflammation (Tomova et al., 2015). Probiotics have been shown to reduce gut inflammation through numerous mechanisms including lowering gut barrier permeability, decreasing inflammatory cytokines, and other immunomodulatory effects. In pregnant female mice, maternal immune activation (MIA) results in impaired intestinal barrier integrity and symptoms like autism in the offspring, which are related to microbiome dysbiosis (Hsiao et al., 2013). After Bacteroidetes fragilis treatment, the repetitive behaviors were attenuated and intestinal permeability was restored, and the gut microbiota imbalance partially improved in the offspring (Hsiao et al., 2013). The probiotic mixture VSL#3 significantly improved sociability, social interaction, anxiety-liked behavior, and behavioral despair, while reversing the increase in serum IL-6 and decrease in serum IL-10 induced by prenatal VPA exposure (Adıgüzel et al., 2022). Moreover, daily Lactobacillus strain supplementation supports gut–brain axis in the VPA-induced prenatal model of autism by reversing autistic deficits and improving immune functions (Sunand et al., 2020). In their study, treatment with Lactobacillus strains decreased TNF-α levels in serum and IL-6 in the brain. TNF-and IL-1 attach to the brain’s endothelial cells to trigger immunological responses in the brain (de Theije et al., 2011). In addition, reduced IL-6 levels have been shown to enhance GABAergic interneuron activity, which in turn increases GAD65/67 levels, preventing the loss of parvalbumin-positive (PV⁺) neurons and GABA levels (Basta-Kaim et al., 2015).