Gut bacteria traits and effects of fecal transplant treatment in children with autism in central China

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition that originates in infancy and early childhood. Its core characteristics include persistent deficits in social communication and interaction, as well as restricted interests and repetitive patterns of behavior (1).In recent years, the prevalence of ASD has shown a consistent upward trend. According to data from the Centers for Disease Control and Prevention (CDC), the prevalence of ASD among 8-year-old children rose from 2.27% in 2018 to 2.7% in 2023 (2, 3). National epidemiological surveys in China similarly reported a prevalence of 0.7% among school-aged children (ages 6–12) in 2020 (4), whereas young children (0–6 years) showed a prevalence of 1.8% in 2023 (5). Conservatively estimated, approximately 2 million children aged 0–14 years are currently affected by ASD in China, with 160,000 new cases emerging annually (6). The high disability rate and incurability of ASD have created a significant public health burden.

The pathogenesis of ASD remains incompletely elucidated. Current evidence suggests that its etiology involves an interplay of genetic susceptibility, immune dysregulation, and environmental factors (7).At present, primary interventions predominantly focus on behavioral therapy and educational support, with a significant lack of specific agents aimed at addressing core symptoms (8, 9). Significantly, children with ASD exhibit higher susceptibility to comorbidities-including functional gastrointestinal disorders (e.g., abdominal pain, constipation, diarrhea), sleep disturbances, and emotional/behavioral problems-compared to neurotypically developing (TD) children (10 –12). These comorbidities not only substantially impair quality of life but may also exacerbate core behavioral symptoms (13, 14). Consequently, investigating the underlying pathophysiological mechanisms and developing novel interventions are key priorities in research on neurodevelopmental disorders.

In recent years, advances in the gut-brain axis (GBA) theory have intensified investigations into the mechanistic roles of gut microbiota in neurodevelopment. This theory highlights the bidirectional communication between gut microbiota and the central nervous system through multiple pathways including chemical, neural, immune, and endocrine routes, which ultimately modulate brain development and function (15 –17). The pathological significance of this axis is now widely recognized, as mounting evidence implicates intestinal dysbiosis as a crucial susceptibility factor in the progression of numerous neurological disorders, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and notably, ASD (18). In the context of ASD specifically, a consistent body of evidence points to characteristic dysbiosis. The main manifestations are featuring reduced microbial diversity, depletion of beneficial bacteria [including short-chain fatty acid (SCFA)-producing taxa], and enrichment of potential pathogens such as the Clostridium genus (19 –27). In addition, metabolomic investigations further demonstrate microbial involvement in ASD pathogenesis via dysregulation of key metabolic pathways, including SCFA production, tryptophan metabolism, and phenolic compound synthesis (25, 28 –35). Crucially, this dysbiosis is now understood to be a potent driver of immune dysregulation, a core pathological feature in ASD. Emerging studies highlight how an imbalanced gut microbiome can trigger pro-inflammatory responses and compromise intestinal barrier integrity, contributing to the neuroinflammatory states observed in the disorder (36). However, certain heterogeneity persists across studies due to variations in geographic distribution, dietary patterns, antibiotic exposure history, and subtypes of ASD among the cohorts (20, 37 –39). Despite this variability, the compelling link between the gut microbiome and ASD pathophysiology has spurred the exploration of microbiota-targeted therapies. This approach is built upon the growing success of such interventions in managing other conditions rooted in microbial dysbiosis. A recent comprehensive review, for example, highlights the therapeutic potential of modulating the gut microbiome for a range of gastrointestinal disorders, thereby establishing a strong rationale for exploring these strategies in neurodevelopmental conditions that frequently present with GI comorbidities (40).

FMT is an emerging intervention that restores intestinal microbial balance and has demonstrated potential in autism spectrum disorder (ASD) management. Preliminary evidence indicates FMT ameliorates gastrointestinal symptoms and partially improves core behavioral manifestations in ASD. In an open-label trial administering microbiota transfer therapy (MTT) to 18 children with ASD for a period of 10 weeks, over 80% experienced relief from gastrointestinal symptoms such as abdominal distension and diarrhea, and the symptom improvement could last for up to 2 years (41, 42). Furthermore, a recent study reported 20% and 17% reductions in Autism Behavior Checklist (ABC) and Social Responsiveness Scale (SRS) scores, respectively, among 40 children following a 4-week FMT protocol (43). Although FMT has shown preliminary therapeutic efficacy in treating ASD, its clinical application encounters significant challenges. Specifically, the lack of standardized protocols for the preparation of fecal bacterial liquid, administration routes and treatment plans has hindered the wide implementation of this treatment measure (44). More importantly, most current studies utilize microbiota from adult donors. However, the developing gut ecosystem in children may display age-related incompatibilities in microbial composition and metabolic functions when compared to adult-derived communities. This discrepancy could potentially lead to immune dysregulation and disorders related to sexual development (45, 46). Currently, there is still a lack of a systematically constructed child-specific donor microbiota bank, which has become one of the main limiting factors for the application of FMT in the pediatric ASD population. Therefore, exploring the pediatric donor microbiota with clearly defined sources and age-matched is anticipated to offer a more suitable microecological foundation for the safety and efficacy of FMT in pediatric ASD.

In light of the aforementioned context, this research is structured into two distinct components. The first component consists of a cross-sectional cohort study designed to systematically assess the differences in intestinal microbiota characteristics between children diagnosed with ASD and healthy control children in the central region of China. The second component involves a single-center, self-controlled open-label study that will administer FMT via colonoscopy, utilizing microbiota from pediatric donors. This intervention seeks to evaluate the relationship between clinical improvements and behavioral symptomatology in children with ASD, both prior to and following the treatment. Collectively, this study aims to establish a foundational understanding of the mechanisms underlying FMT, enhance the development of individualized treatment strategies, and establish a safety framework for the application of FMT in pediatric populations.

This study employed a streamlined FMT protocol consisting of three days of bowel preparation followed by six days of transplantation. The intervention resulted in notable improvements in core behavioral symptoms, as evidenced by a 14.6% reduction in CARS scores, and a marked alleviation of gastrointestinal (GI) comorbidities in children with ASD. Interestingly, the changes in the ABC scores did not reach a statistically significance. We hypothesize that the lack of statistical significance could be partly attributed to the limited statistical power resulting from our small sample size. In addition, as a parent-reported checklist, the ABC is inherently subjective, which may introduce variability and affect the sensitivity of the measurement in detecting subtle behavioral changes over the study period. Additionally, significant alterations in gut microbiota composition were observed.

Microbiota diversity analysis revealed distinct dysbiotic features in the ASD group, including a significant reduction in α-diversity and clear separation in β-diversity compared to healthy controls—findings consistent with prior reports by Kang and Ding et al (51, 52). Following the FMT treatment, the microbial profiles of ASD children gradually shifted toward those of healthy donors. This shift was characterized by increased relative abundances of Firmicutes and Bacteroidetes and decreased Proteobacteria at the phylum level. Increased Ruminococcaceae and Lachnospiraceae, and decreased Enterobacteriaceae at the family level. And increased Prevotella, Faecalibacterium, Agathobacter, and Dorea, with a reduction in Escherichia-Shigella at the genus level. These compositional changes were correlated with clinical improvements.

Among these, Prevotella—a key genus involved in dietary fiber degradation and vitamin B1 metabolism—showed a positive correlation between its post-FMT increase and improved social behaviors (22). Faecalibacterium, particularly F. prausnitzii, known for its butyrate production and anti-inflammatory properties, was associated with relief of constipation symptoms, corroborating findings by He et al. (53). The genus Dorea may also contribute to maintaining mucosal barrier integrity by modulating Treg/Th17 cell balance (54, 55). Furthermore, the observed reduction in Escherichia-Shigella may help ameliorate neurotransmitter imbalances by decreasing aberrant gamma-aminobutyric acid (GABA) synthesis—a phenomenon supported by previous studies demonstrating GABA normalization following FMT (43, 56).

Interestingly, the post-FMT decrease in Bifidobacterium abundance contradicted expectations, as this genus is generally considered beneficial. This unexpected finding may be explained by the "distant attack, close defense" colonization model proposed by Qin et al (57). According to this model, close phylogenetic similarity between donor and recipient strains—such as in this study using pediatric donors and pediatric recipients—may lead to niche competition and colonization resistance, thereby hindering donor strain establishment. This highlights the need for future research to incorporate metagenomic analyses of donor-recipient strain compatibility to optimize FMT outcomes.

The dynamics of Agathobacter abundance revealed a paradoxical pattern. Specifically, its abundance demonstrates a positive correlation with the scores on the ABC and CARS scales. However, following fecal microbiota transplantation (FMT) treatment, there is an observed increase in the abundance of this genus, yet a significant reduction in the scale scores occurs. This apparent contradiction may reflect the genus's complex metabolic capacity. While Agathobacter is a known butyrate producer with anti-inflammatory effects, some strains can also generate metabolites such as succinate, which modulates immunity (58, 59). In the ASD gut environment, succinate may stimulate serotonin (5-HT) production by enterochromaffin cells. Elevated peripheral 5-HT levels have been positively associated with stereotyped behaviors in ASD (60, 61). Furthermore, genetic ablation of intestinal 5-HT synthesis in animal models has been shown to significantly reduce autism-like behaviors (62). Thus, the net clinical effect of Agathobacter may depend on the balance between its beneficial metabolites (e.g., butyrate) and its neuroactive byproducts (e.g., those promoting 5-HT synthesis).

In summary, this study demonstrates that a simplified FMT protocol can effectively alleviate both core behavioral symptoms (CARS scores) and GI comorbidities in children with ASD, with 100% of participants reporting GI symptom improvement. These findings provide preliminary clinical evidence supporting the therapeutic potential of FMT in this population.

However, several limitations must be acknowledged. First, the sample size was relatively small, which may limit the statistical power and generalizability of the findings. Second, the behavioral and GI assessment tools used lacked standardization. Future studies should incorporate internationally validated instruments, such as the Gastrointestinal Symptom Rating Scale (GSRS), Children's Sleep Habits Questionnaire (CSHQ), and Social Responsiveness Scale (SRS), to enhance comparability and objectivity. Third, the study did not include metabolomic analyses, precluding direct validation of gut–brain axis mechanisms through key microbial metabolites such as butyrate and 5-HT. Besides, due to the lack of a double-blind, randomized controlled trial (RCT) makes it difficult to definitively confirm the therapeutic efficacy of FMT and to rule out potential placebo effects. Lastly, although pediatric donors were used, the absence of an adult donor control group limited the ability to directly assess age-specific microbial advantages. Future research should expand sample sizes, integrate metagenomics and metabolomics approaches, and further elucidate microbiota–gut–brain interactions. Randomized controlled trials should also explore the clinical value of age-matched pediatric donor microbiota, thereby laying the groundwork for developing a dedicated pediatric microbiota donor bank.

This study provides preliminary evidence that a simplified pediatric FMT protocol can improve behavioral symptoms and gastrointestinal comorbidities in children with ASD. The intervention led to measurable changes in gut microbiota composition, some of which were positively correlated with symptom improvement. Key taxa such as Prevotella, Faecalibacterium, and Dorea may contribute to therapeutic effects via immune modulation, barrier maintenance, and neurotransmitter regulation. The results underscore the potential of age-matched donor microbiota in enhancing colonization compatibility and clinical efficacy. However, larger-scale, multi-omics studies and randomized controlled trials are needed to confirm these findings and inform the development of safe, standardized pediatric FMT protocols.