**3. Restoring the gut ecosystem: therapeutic outlooks for autism spectrum disorder**

Despite increased ASD diagnoses, there remains no US Food and Drug Administration (FDA)-approved pharmaceutical treatment to alleviate core symptoms of ASD [69]. Currently, recommended management strategies essentially involve rehabilitation, educational interventions, speech therapies, psychiatric medications, and specific treatments for individual comorbidities [70], all with limited success [71]. Considered the emerging role of gut dysbiosis in ASD, interest in rebalancing human GM as a possible therapeutic approach is growing [72]. Indeed, targeting the GM in children with ASD through administration of ATBs, pro- and prebiotics, and nutritional approaches or, more recently, through fecal microbiota

*Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*

producing anti-inflammation butyrate [45].

Thus, the existence of a GI dysbiosis as an actor in the ASD etiopathogenesis remains a controversial topic. Indeed, other studies comparing children with ASD and their healthy siblings reported no meaningful difference in GM composition [53, 54]. According to the authors, other explanations for the GI dysfunction in this population should be considered, including elevated levels of anxiety and self-restricted diets. Therefore, given the higher incidence of ATB usage and often different diets compared with neurotypical individuals, both of which can alter the

Dysbiosis in ASD involves not only bacterial species but also yeasts, as reported in recent studies [40, 41, 55, 56]. One culture-based study showed significant presence of *Candida* species in the feces of children with ASD, mainly *Candida albicans*.

It also identified hyphae formation, suggesting that the dimorphic yeast had switched to its invasive and adhesive form. However, another study did not report such overrepresentation of *Candida* in ASD children compared to control ones [49]. Moreover, correlations between ASD and GI disturbances may not alone be driven by the composition of the GM but also by differences in its functionality, such as the bacterial metabolites that could play a role in the GBA. Indeed, overproduction of

composition of the GM, such data should be interpreted with care.

compositions of GM compared to healthy controls [36–43]. Moreover, exciting work with animal models widely deepened the possible role of gut microorganisms in the pathogenesis of such disorders [44, 45]. These evidences have led to the hypothesis that GM alteration is not only associated with ASD but may play a key role in the exacerbation of ASD symptoms and/or its pathogenesis, at least for some ASD subgroups [14]. Overall, most studies agree that GM composition is distinctive in ASD compared to healthy controls, but results are often inconsistent as to the nature and/or extent of GI bacterial community differences, failing to generate a coherent picture. Microbiota analyses reported tenfold higher counts of pathogenic *Clostridium* spp. in children with ASD compared with healthy controls [46, 47]. *Clostridium* is known to produce neurotoxins and P-cresol, cause higher propionic acid levels, and promote conditions that favor inflammation and exacerbate ASD symptoms. On the other hand, there have been some consistent findings of decrease in certain beneficial bacteria, specifically *Bifidobacterium* [40, 48, 49] known for its health-benefit properties. Then, the composition of GM of ASD individuals has been characterized, showing a reduction of the *Bacteroidetes*/*Firmicutes* ratio which pointed to elevated numbers of *Firmicutes* in contrast to decreased levels of *Bacteroidetes* [36, 39, 41, 49–51]. *Bacteroidetes* are short-chain fatty acids (SCFA) producing bacteria, and their metabolites, especially propionic acid, may influence the CNS and autism behavior by modulating the GBA [36]. Moreover, species of *Desulfovibrio* were also isolated from the stool of patients with ASD, and, to a lesser extent, in non-affected siblings [52]. *Desulfovibrio* could be an important contributor to GI inflammation, as its major metabolic by-product—hydrogen sulfide—is cytotoxic to colonic epithelial cells. Furthermore, the presence of autistic symptoms in children with ASD has been correlated with a less diverse gut microbiome, with less carbohydrate degrading and fermenting bacteria of the genera *Prevotella*, *Coprococcus*, and the unclassified *Veillonellaceae* in ASD microflora samples as compared to the healthy controls [39]. This decrease in GM diversity can lead to a loss of key signals required for normal brain maturation. Additionally, increased *Sutterella* were found in significant numbers in intestinal biopsies and stools of ASD children [48, 50]. This genus is known to regulate mucosal metabolism and intestinal epithelial integrity. Lastly, there were still some conflicting results about the alterations of *Akkermansia*, *Ruminococcus*, and *Faecalibacterium* in ASD patients. *Akkermansia* and *Ruminococcus* are mucin-degrading bacteria [40], and *Faecalibacterium* is regarded as commensal or even beneficial due to its function of

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transplantation (FMT) has been shown to improve not only GI disturbances but also behavioral and neurophysiological abnormalities associated with ASD [18, 24].
