Probiotic application in autism treatment: a systematic review

Introduction

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition characterized by impairments in social communication and social interaction, as well as restricted and repetitive patterns of behavior (DSM-5 Diagnostic Classification 2013). Symptoms can be observed in children as early as the first year of life and are often associated with lifelong challenges (Tan et al. 2021). In addition to autism-specific developmental impairments, a higher prevalence of other medical conditions has been reported, including gastrointestinal (GI) symptoms, seizures, anxiety, sleep disturbances, and immune system dysfunctions (Hossain et al. 2020; Kohane et al. 2012). GI symptoms, in particular, such as diarrhea, constipation, and abdominal pain, are commonly reported in children with ASD. An increasing number of studies suggest that comorbid GI conditions may have secondary effects on problematic behaviors observed in ASD (Ferguson et al. 2019). While the relationship between the gut and the brain has long been hypothesized, recent studies have begun to explore gut-brain interactions within the context of ASD. These studies have revealed connections between gut microbiota and ASD pathophysiology (Cryan et al. 2019).
Studies have shown that probiotics and prebiotics have positive effects on GI symptoms and other symptoms associated with ASD (Arnold et al. 2019; Sanctuary et al. 2019; Wang Y. et al. 2020). Probiotic supplementation aids in restoring the microbiota, thereby improving ASD-related symptoms. Various studies have demonstrated the positive effects of probiotic consumption on GI symptoms and behavior (Slykerman et al. 2018). The imbalance in the composition of the gut microbiota and microbial metabolites, such as short-chain fatty acids (SCFAs), in children with ASD has been shown to contribute to the etiology of ASD (MacFabe 2015; Wang L. et al. 2012). SCFAs, including butyrate, propionate, and acetate, are produced by gut bacteria through fermentation (Wang L. et al. 2012). These fatty acids are vital modulators of the microbiota-gut-brain axis, regulating behavior and gut physiology (Wang Y. et al. 2020). Butyrate serves as an energy substrate, inhibits histone deacetylases, exerts anti-inflammatory effects, and regulates blood-brain barrier and gut permeability (Wang Y. et al. 2020). Studies in rodents have shown that behaviors resembling ASD, such as repetitive behaviors and object preferences, are observed when propionic acid is injected peripherally or centrally (Wang L. et al. 2012). Acetate, along with butyrate and propionate to a lesser extent, enhances colonic blood flow, influencing muscle contraction, tissue oxygenation, and nutrient supply (Wang Y. et al. 2020). We hypothesized that probiotic supplementation might improve SCFA abnormalities in the intestines of children with ASD. SCFAs can cross the blood–brain barrier and modulate serotonin (5-hydroxytryptamine, 5-HT) and dopamine production, which could affect early brain development (Carlessi et al. 2021; De Angelis et al. 2013). Alterations in the gut microbiota may lead to neurotransmitter dysregulation in the peripheral system and neurodevelopmental disorders (Morris et al. 2017). Beneficial bacteria such as Lactobacillus, Bifidobacteria, and Bacillus contribute to the production of several important neurotransmitters, including dopamine, norepinephrine, serotonin, gamma-aminobutyric acid (GABA), acetylcholine, and histamine (Luna et al. 2017; Morris et al. 2017). Probiotic consumption is thought to help improve neurotransmitter dysregulation. This study was conducted to explore the potential effects of probiotic consumption in individuals with autism spectrum disorder.

Methods

Articles included in the study were comprehensively searched in electronic databases such as PubMed, Scopus and Google Scholar. Search terms were defined as “autism,” “probiotics,” and combinations of these terms. Filters were applied to include studies published from the inception of the databases to the present. Book chapters, descriptive studies, conference proceedings, letters to the editor, literature reviews, narrative reviews, systematic reviews with or without meta-analysis, and nutritional adjunctive intervention studies were excluded (Fig. 1).
Table 1 shows the potential effects of probiotics on ASD. Additionally, Table 1 also highlights the potential side effects that may result from following these probiotic practices.

Bacterial CFU count of probiotics

The total bacterial count used in the studies varies. Two studies included probiotics containing 100 × 108 CFU of bacteria, while three studies did not specify the bacterial count. In the other studies, probiotics with different bacterial counts were used (Fig. 2).

Evidence in specific syndromes

In a 108-day study conducted by Wang Y. et al. with 50 children with ASD, probiotic supplementation containing the strains Bifidobacterium infantis Bi-26, Lactobacillus rhamnosus HN001, Bifidobacterium lactis BL-04, and Lactobacillus paracasei LPC-37 resulted in an overall improvement in ASD symptoms. Additionally, improvements were observed in gastrointestinal symptoms, hyper-serotonergic states, and impaired dopamine metabolism. The study also reported increases in Bifidobacteriales and Bifidobacterium longum and decreases in Clostridium counts in the children’s gut microbiota. Furthermore, increases in levels of short-chain fatty acids, such as acetic acid, propionic acid, and butyric acid, were noted in the intestines of individuals with ASD (Wang Y. et al. 2020). In a separate study, ten children with ASD, aged three to twelve years, were given a probiotic supplement containing the following strains: Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve. The results showed improvements in microbiota composition and diversity, with increased Lactobacillus counts in the gut and reduced gastrointestinal symptoms (Arnold et al. 2019). In another study involving an 18-week probiotic intervention, a reduction in gastrointestinal symptoms, including constipation, diarrhea, indigestion, and abdominal pain, was observed in individuals with ASD. Behavioral improvements were also reported, along with an increase in the number of Bifidobacterium, Prevotella, and Desulfovibrio bacteria in the gut (Kang et al. 2017). A study by Santocchi et al. demonstrated that probiotic administration resulted in a reduction of gastrointestinal symptoms, improved intestinal inflammation, and a reduction in behavioral disturbances, such as irritability, anger, aggressive behavior, and sleep disorders (Santocchi et al. 2016). In an 18-week study involving 18 children and adolescents with ASD, probiotic supplementation resulted in a decrease in gastrointestinal symptoms such as constipation, diarrhea, indigestion, and abdominal pain, as well as behavioral improvements. Additionally, increases in gut bacterial diversity and in the number of bacteria such as Bifidobacteria and Prevotella were observed (Kang et al. 2019). In a study conducted on 160 children with ASD aged between 6 and 12 years, it was reported that probiotic administration containing Lactobacillus strains resulted in a decrease in gastrointestinal symptoms and an increase in intestinal bacterial diversity and Bifidobacteria count (Zhang et al. 2022a). Lastly, a study involving 160 children with ASD, aged 6-12 years, reported that probiotic supplementation containing Lactobacillus strains led to a reduction in gastrointestinal symptoms, increased gut bacterial diversity, and an increase in the number of Bifidobacteria (Kong et al. 2021). In a study involving a probiotic supplement containing Bifidobacterium and BCP group strains, a reduction in gastrointestinal symptoms and improvements in atypical and abnormal behaviors were observed. Additionally, a decrease in the inflammatory cytokine levels of interleukin (IL)-13 and tumor necrosis factor α (TNF-α) was noted (Sanctuary et al. 2019). Another study reported a reduction in hyperactivity levels in individuals with autism following probiotic administration. Furthermore, improvements in impulsivity symptoms, CPT scores, and quality of life scores were noted (Rojo-Marticella et al. 2025). In a study by Grimaldi et al. on 30 individuals with autism, probiotic supplementation resulted in improvements in gastrointestinal symptoms, such as abdominal pain and bowel movements, with a reduction in Bifidobacterium spp., Veillonellaceae levels, and an increase in Faecalibacterium prausnitzii, Lachnospiraceae, and Bacteroides spp. levels. Improvement in antisocial behaviors was also observed (Grimaldi et al. 2018). In a study conducted by Sherman et al., probiotic supplementation led to a reduction in serum anti-tubulin, serum CaM kinase II, serum anti-dopamine receptor D1, and serum SpCO levels (Sherman et al. 2022). Another study noted reductions in autism severity, restricted/repetitive behaviors, improvements in social functioning, microbiome composition, and immune profile after probiotic administration (Mazzone et al. 2024). In a study with 71 individuals with ASD, aged 7 to 15 years, supplementation with Lactobacillus plantarum led to increased scores on the Social Responsiveness Scale, the Swanson, Nolan, and Pelham IV-Taiwan version, and the Child Behavior Checklist, as well as reductions in oppositional and stubborn behaviors (Liu et al. 2019). Another study reported that administering probiotics containing Streptococcus, Bifidobacterium, and Lactobacillus strains resulted in reduced beta and gamma band power in the frontopolar regions, increased coherence in the beta and gamma bands, a shift in frontal asymmetry, and improvements in brain activity (Billeci et al. 2023). In a study by Narula Khanna et al., probiotic supplementation resulted in improvements in behavior, a decrease in the severity of behavioral symptoms as measured by the SRS-2, and reductions in social withdrawal, lethargy, stereotypic behaviors, hyperactivity, inappropriate speech, gastrointestinal symptoms, constipation, and diarrhea (Narula Khanna et al. 2025). Yang et al. (2023) reported that in their study of both male and female individuals with ASD, probiotic administration led to reductions in pro-inflammatory sICAM-1 and sVCAM-1 levels in adults. Additionally, in children, reductions in sICAM-1, sVCAM-1, IL-12, IL-23p40, and IL-2Rα levels, along with increases in formic, acetic, and propionic acid levels, were observed (Yang et al. 2023). In a study by Schmitt et al., probiotic supplementation led to improvements in Vineland-3 scores, adaptive behaviors, orientation, cognitive behaviors, behavioral test scores, behavior composite scores, social/geometric imagery ratios, and eye tracking (Schmitt et al. 2023). Probiotic supplementation containing Lactobacillus rhamnosus strain GG and Bifidobacterium animalis subsp. lactis Bb12 led to reductions in gastrointestinal comorbidities and psychiatric symptoms in individuals with ASD. In male participants, a reduction in intestinal Candida albicans density and no change in Saccharomyces cerevisiae density was observed. Additionally, improvements in Candida albicans antibody levels and gut disturbances associated with Candida albicans were noted. No changes in Candida albicans or Saccharomyces cerevisiae density were reported in female participants (Severance et al. 2017). In various studies, probiotic supplementation in individuals with autism has been reported to result in a reduction in gastrointestinal symptoms such as diarrhea and constipation, an increase in bowel movement frequency from once every 1.5 days to once every 1.3 days, a decrease in Autism Treatment Evaluation Checklist (ATEC) scores, improvements in speech, language, and communication skills, increased socialization, enhanced sensory/cognitive awareness, behavioral improvements, increased levels of Bifidobacteria and Lactobacilli in the gut, a decrease in body weight, a reduction in Clostridium XIVa levels, and an increase in behavior scale scores (Kałużna-Czaplińska and Błaszczyk 2012; Parracho et al. 2010; Shaaban et al. 2018; West 2013; Zhang et al. 2022b).

Effects on behavior

Various studies have reported behavioral improvements following probiotic use at different doses. These improvements include reduced irritability, anger, and aggression; decreased sleep disturbances; better scores on the Social Sensitivity Scale; improved Abnormal Behavior Checklist scores; better Clinical Global Impression Scale scores; enhanced social cognition responses; reductions in atypical and abnormal behaviors; decreased hyperactivity; improvements in impulsivity symptoms; and anti-social behavior improvement, reduced restricted/repetitive behaviors, enhanced social functionality, increased Child Behavior Checklist scores, better Swanson, Nolan, and Pelham-IV-Taiwan version scores, decreased oppositional and stubborn behaviors, reduced social withdrawal, improved lethargic behaviors, reduced stereotypical behaviors, improved speech, language, and communication skills, and increased social interaction, sensory/cognitive awareness, and behavior scale scores (Grimaldi et al. 2018; Ka³u¿na-Czapliñska and B³aszczyk 2012; Kang et al. 2017; Kong et al. 2021; Liu et al. 2019; Mazzone et al. 2024; Narula Khanna et al. 2025; Parracho et al. 2010; Rojo-Marticella et al. 2025; Sanctuary et al. 2019; Santocchi et al. 2016; Schmitt et al. 2023; West 2013; Zhang et al. 2022b).

Adverse effects

Figure 3 shows the side effects reported in the studies. Four studies reported no adverse effects in individuals with autism spectrum disorder following probiotic supplementation: Kang et al. (2019), Liu et al. (2019), Narula Khanna et al. (2025), and Wang Y. et al. (2020). One study reported nausea and skin rash as side effects in a few ASD individuals who received Lactobacillus plantarum supplementation (Parracho et al. 2010). Another study by Shaaban et al. found that probiotics, when added to diets, resulted in side effects such as diarrhea, bloating, abdominal cramps, and skin rashes in some participants (Shaaban et al. 2018). A four-week study of male and female individuals with ASD reported temporary side effects, including stereotypies, fever, hyperactivity, reflux, abdominal pain, nausea, and bloating, in some participants who took probiotics containing the L. reuteri strain (Mazzone et al. 2024).
In a different study by Arnold et al. (2019), abdominal distension, flatulence, nausea, vomiting, and depressed mood were observed as side effects. Similarly, other studies reported mild to moderate hyperactivity, tantrums, aggression, increased gassiness, stomachaches, sleep disturbances, and lethargy as side effects following probiotic supplementation (Kang et al. 2017; Sanctuary et al. 2019).

Conclusions

Probiotics appear to have significant, positive effects on children with autism spectrum disorder. A study of 1,374 children, adolescents, and adults from 24 different studies found that probiotics had positive therapeutic effects on behavior, gastrointestinal symptoms, and other symptoms in children with ASD. The study found that probiotic use positively affected behavior and gastrointestinal and other symptoms in children with ASD. Furthermore, subgroup analyses revealed that studies with longer intervention periods and randomized controlled trial designs were more likely to demonstrate the impact of probiotic treatment on behavioral symptoms associated with ASD. To better demonstrate the therapeutic effects of probiotics on individuals with autism, randomized, double-blind, placebo-controlled trials conducted in accordance with strict clinical trial guidelines are needed.

Disclosures

This research received no external funding.
Institutional review board statement: Not applicable.
The authors declare no conflict of interest.

References