**2.2 Disruptions of microbial colonization in autism spectrum disorder**

Imbalances in GM composition at the first stages of life and concomitant behavioral changes have been related to various prenatal and early-life events [8].

Mode of birth, whether through natural birthing process or Cesarean section (C-section), greatly affects the initial microbial settlement. Vaginally delivered babies harbor bacteria similar to their mothers' fecal and vaginal microbiota with dominance of *Bifidobacterium,* whereas babies born via C-section would acquire an altered GM composition resembling to their mother's skin microbiota as well as microbes from the surrounding environment with a delayed colonization by bacteria from maternal origin, mainly enterobacteria, bifidobacteria, and *Bacteroides* [11]. Interestingly, these modifications can persist for several months [2]. Thus, concern has arisen that this reduced microbial diversity and altered bacterial profile in babies delivered via C-section could contribute to ASD. Indeed, studies employing animal models have revealed that, compared with vaginally delivered animals, those delivered by C-section suffer more frequently from behavioral abnormalities that have been associated with ASD [19]. Consistent with this, some clinical studies report that C-section births in comparison to natural vaginal delivery are associated with a significantly 23% higher risk of the child developing ASD [20]. Thus, the importance of C-section in conditioning negative effects on the CNS is debated.

In terms of gestational age at birth, preterm neonates (PT) are characterized by a delayed microbial colonization, missed or reduced acquisition of *Lactobacillus* and *Bifidobacterium*, greater abundance of *Proteobacteria*, and earlier acquisition of *Firmicutes* [11], hence increasing the risk to develop later disease. One study reported a relationship between premature birth and an increase risk of ASD development [21]. Interestingly, each week of shorter gestation was associated with an increased risk of ASD. The early development of infant at birth has also been recognized as a biomarker of future risk of neurodevelopmental disorders. Besides, gestational growth affects the neurodevelopment, and both infants born small or large for gestational age are associated with higher rates of ASD [22].

Early feeding pattern also interferes greatly in the regulation of the intestinal colonization, with breastfed infants harboring a less diverse but more stable and uniform GM. Specific biological markers of healthy GM, including early colonization of *Bifidobacterium* and greater prevalence of *Lactobacillus*, characterize microbial communities of breastfed infants [11]. By contrast, increased species richness accompanied by an overrepresentation of *Clostridium* prevails in bottlefed ones. In fact, cognitive functioning and neurological development of children have been associated with the duration of exclusive breast-feeding and introduction of formula feed [23]. Remarkably, breast-feeding appears to be less frequent and, when present, occurs for a much shorter duration in children with ASD [24]. Further differentiation occurs after the introduction of solid foods. Feeding patterns and strong food preferences of ASD children for nutrient-poor starchy foods while rejecting fruits, vegetables, and proteins may lay the foundation of an abnormal GM. The high prevalence of ASD in some countries has been correlated with typical carbohydrate rich diet and consequently predominance of genera *Prevotella* and *Megasphaera* [25]. Therefore, understanding how these changes affect human GM suggests that early dietary habits have a more complex effect on the metabolic programming of a child than previously anticipated.

**173**

*Could Gut Modulation through Probiotic Supplementation Be Beneficial in Autism Spectrum…*

Additional modifications are induced by exposure to drugs either directly or indirectly from the mother. ATB-induced shifts in the gut microbial composition can persist several months after cessation of the treatment, inducing long-term dysbiosis [26]. An association between long-term ATB use, hospitalization, abdominal discomfort, and the onset of ASD symptoms has been shown [27]. Likewise, a population-based cohort study revealed the use of various ATBs during pregnancy as a potential risk factor for ASD [28]. Using animal models, periconceptional exposure to nonabsorbable ATB was shown to induce variations in GM composition in offspring associated to reduction in social interactions and increased anxiety [29]. Other drugs lead to similar observations. In mice, modeling maternal exposure to valproic acid, an antiepileptic drug induced lasting changes in the offspring GM composition, which was associated with neuroinflammation, abnormal GI physiology, and ASD-like behavioral abnormalities [30]. All these observations point to the common hypothesis that early exposure to drugs can modify GM composition transiently or permanently, possibly affecting the severity of non-GI-related

Apart from exposition to drugs, another emerging explanation for the difference in GM between ASD and healthy individuals is immunological. Maternal infection during pregnancy was shown to alter microbial composition and is a primary environmental risk factor for ASD [31]. GM alterations (especially in the bacterial classes clostridia and bacteroidia) and higher gut permeability are seen in a maternal immune activation model of ASD [18]. Male progenies of pregnant mice injected during pregnancy with a viral mimetic develop abnormal communication and sociability, repetitive behaviors, and increased anxiety. Another well-established brain-gut connection is the role of stress and its mediators in altering the GM. Maternal separation is a typical model of early-life stress employed in animal studies. In rats, early maternal separation does not only lead to a dysbiotic state of the GM that persists into adulthood but also results in functional GI symptoms and long-term cognitive and behavioral deficits [32]. Likewise, psychological stress was shown to alter colonic mucosa-associated microbiota, with significant decrease in abundance of health-benefit bacteria, such as *Lactobacillus*, while abundance of clostridia increased [33]. Although one cannot extrapolate from mice to human without further evidence, these findings nonetheless suggest that the differences seen in the GM of ASD patients may be a result of immunological changes.

Further, metals and other contaminants have also been identified to increase the risk for ASD knowing their capacity to interfere with the composition and metabolic activity of the GM. In fact, results of a study evaluating the interaction between environmental chemicals and GI microorganisms suggest that alterations in the levels of seven elements (Pb, As, Cu, Zn, Mg, Ca, and Hg) and nine genera of GM (*Bacteroides*, *Parabacteroides*, *Sutterella*, *Lachnospira*, *Bacillus*, *Bilophila*, *Lactococcus*, *Lachnobacterium*, and *Oscillospira*) may be related to ASD [34]. Excessive accumulation of these typical neurotoxic elements leads to abnormally increased abundance of several genera and is reported to be closely related to poorer

Taken together, ASD is thought to be a result of a combination of different

Previously, much research effort on ASD focused on genetic, neurological, and behavioral aspects of disease. Recently, the evidence of the impact of dysbiosis on CNS raised interest in the analysis of the potential link between GM and ASD. So far, various studies demonstrated that children with ASD exhibit different

interacting mechanisms that each contributes a fraction of disease risk.

**2.3 Microbial and metabolic dysregulations in autism spectrum disorder**

*DOI: http://dx.doi.org/10.5772/intechopen.89375*

symptoms of ASD patients.

intellectual function [35].

#### *Could Gut Modulation through Probiotic Supplementation Be Beneficial in Autism Spectrum… DOI: http://dx.doi.org/10.5772/intechopen.89375*

Additional modifications are induced by exposure to drugs either directly or indirectly from the mother. ATB-induced shifts in the gut microbial composition can persist several months after cessation of the treatment, inducing long-term dysbiosis [26]. An association between long-term ATB use, hospitalization, abdominal discomfort, and the onset of ASD symptoms has been shown [27]. Likewise, a population-based cohort study revealed the use of various ATBs during pregnancy as a potential risk factor for ASD [28]. Using animal models, periconceptional exposure to nonabsorbable ATB was shown to induce variations in GM composition in offspring associated to reduction in social interactions and increased anxiety [29]. Other drugs lead to similar observations. In mice, modeling maternal exposure to valproic acid, an antiepileptic drug induced lasting changes in the offspring GM composition, which was associated with neuroinflammation, abnormal GI physiology, and ASD-like behavioral abnormalities [30]. All these observations point to the common hypothesis that early exposure to drugs can modify GM composition transiently or permanently, possibly affecting the severity of non-GI-related symptoms of ASD patients.

Apart from exposition to drugs, another emerging explanation for the difference in GM between ASD and healthy individuals is immunological. Maternal infection during pregnancy was shown to alter microbial composition and is a primary environmental risk factor for ASD [31]. GM alterations (especially in the bacterial classes clostridia and bacteroidia) and higher gut permeability are seen in a maternal immune activation model of ASD [18]. Male progenies of pregnant mice injected during pregnancy with a viral mimetic develop abnormal communication and sociability, repetitive behaviors, and increased anxiety. Another well-established brain-gut connection is the role of stress and its mediators in altering the GM. Maternal separation is a typical model of early-life stress employed in animal studies. In rats, early maternal separation does not only lead to a dysbiotic state of the GM that persists into adulthood but also results in functional GI symptoms and long-term cognitive and behavioral deficits [32]. Likewise, psychological stress was shown to alter colonic mucosa-associated microbiota, with significant decrease in abundance of health-benefit bacteria, such as *Lactobacillus*, while abundance of clostridia increased [33]. Although one cannot extrapolate from mice to human without further evidence, these findings nonetheless suggest that the differences seen in the GM of ASD patients may be a result of immunological changes.

Further, metals and other contaminants have also been identified to increase the risk for ASD knowing their capacity to interfere with the composition and metabolic activity of the GM. In fact, results of a study evaluating the interaction between environmental chemicals and GI microorganisms suggest that alterations in the levels of seven elements (Pb, As, Cu, Zn, Mg, Ca, and Hg) and nine genera of GM (*Bacteroides*, *Parabacteroides*, *Sutterella*, *Lachnospira*, *Bacillus*, *Bilophila*, *Lactococcus*, *Lachnobacterium*, and *Oscillospira*) may be related to ASD [34]. Excessive accumulation of these typical neurotoxic elements leads to abnormally increased abundance of several genera and is reported to be closely related to poorer intellectual function [35].

Taken together, ASD is thought to be a result of a combination of different interacting mechanisms that each contributes a fraction of disease risk.

### **2.3 Microbial and metabolic dysregulations in autism spectrum disorder**

Previously, much research effort on ASD focused on genetic, neurological, and behavioral aspects of disease. Recently, the evidence of the impact of dysbiosis on CNS raised interest in the analysis of the potential link between GM and ASD. So far, various studies demonstrated that children with ASD exhibit different

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

the influence of GM in ASD as potential mediator of risk factors.

**2.2 Disruptions of microbial colonization in autism spectrum disorder**

ioral changes have been related to various prenatal and early-life events [8].

large for gestational age are associated with higher rates of ASD [22].

programming of a child than previously anticipated.

Early feeding pattern also interferes greatly in the regulation of the intestinal colonization, with breastfed infants harboring a less diverse but more stable and uniform GM. Specific biological markers of healthy GM, including early colonization of *Bifidobacterium* and greater prevalence of *Lactobacillus*, characterize microbial communities of breastfed infants [11]. By contrast, increased species richness accompanied by an overrepresentation of *Clostridium* prevails in bottlefed ones. In fact, cognitive functioning and neurological development of children have been associated with the duration of exclusive breast-feeding and introduction of formula feed [23]. Remarkably, breast-feeding appears to be less frequent and, when present, occurs for a much shorter duration in children with ASD [24]. Further differentiation occurs after the introduction of solid foods. Feeding patterns and strong food preferences of ASD children for nutrient-poor starchy foods while rejecting fruits, vegetables, and proteins may lay the foundation of an abnormal GM. The high prevalence of ASD in some countries has been correlated with typical carbohydrate rich diet and consequently predominance of genera *Prevotella* and *Megasphaera* [25]. Therefore, understanding how these changes affect human GM suggests that early dietary habits have a more complex effect on the metabolic

protective commensal microbiota along with the overgrowth of pathogenic microorganisms is hypothesized to cause or contribute not only to GI dysfunction but also to ASD-related behavioral symptoms. All these findings have gained an insight into

Imbalances in GM composition at the first stages of life and concomitant behav-

Mode of birth, whether through natural birthing process or Cesarean section (C-section), greatly affects the initial microbial settlement. Vaginally delivered babies harbor bacteria similar to their mothers' fecal and vaginal microbiota with dominance of *Bifidobacterium,* whereas babies born via C-section would acquire an altered GM composition resembling to their mother's skin microbiota as well as microbes from the surrounding environment with a delayed colonization by bacteria from maternal origin, mainly enterobacteria, bifidobacteria, and *Bacteroides* [11]. Interestingly, these modifications can persist for several months [2]. Thus, concern has arisen that this reduced microbial diversity and altered bacterial profile in babies delivered via C-section could contribute to ASD. Indeed, studies employing animal models have revealed that, compared with vaginally delivered animals, those delivered by C-section suffer more frequently from behavioral abnormalities that have been associated with ASD [19]. Consistent with this, some clinical studies report that C-section births in comparison to natural vaginal delivery are associated with a significantly 23% higher risk of the child developing ASD [20]. Thus, the importance of C-section in conditioning negative effects on the CNS is debated. In terms of gestational age at birth, preterm neonates (PT) are characterized by a delayed microbial colonization, missed or reduced acquisition of *Lactobacillus* and *Bifidobacterium*, greater abundance of *Proteobacteria*, and earlier acquisition of *Firmicutes* [11], hence increasing the risk to develop later disease. One study reported a relationship between premature birth and an increase risk of ASD development [21]. Interestingly, each week of shorter gestation was associated with an increased risk of ASD. The early development of infant at birth has also been recognized as a biomarker of future risk of neurodevelopmental disorders. Besides, gestational growth affects the neurodevelopment, and both infants born small or

**172**

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

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

**175**

*Could Gut Modulation through Probiotic Supplementation Be Beneficial in Autism Spectrum…*

Dysbiosis can also affect the functional intestinal barrier that can lead to an alteration in the intestinal permeability referred to as "leaky gut" state, a fundamental factor underlying the relationships between ASD and the GM [63]. Indeed, several reports show increased gut permeability in ASD patients [64]. Disrupted barrier function facilitates the translocation of bacterial metabolites from the gut into the bloodstream to possibly reach the otherwise sterile CNS inducing directly inflammatory reactions. One important bacterial component is the lipopolysaccharide (LPS) that was shown to increase the activity of areas deputed to the emotionalism control such as amygdale [65]. It also leads to the production of inflammatory cytokines that critically alter the physiological brain activity, modulating the neuropeptides synthesis [66]. Moreover, it has been demonstrated that the administration of low doses of LPS in healthy subject is associated to increased pro-inflammatory cytokines and plasma norepinephrine, with higher depression rates, fatigue, and apathy [67]. Consistent with this, a study showed LPS serum levels were significantly higher in autistic patients compared to heath individuals and correlated with

socialization scores in an inverse and independent manner [68].

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

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

bacterial metabolites (e.g., p-cresol) and SCFA (e.g., propionic acid) is frequently described in infants with ASD [30, 49, 57]. These compounds induce intracellular signaling and modulate host gene expression related to neurotransmission systems and behavior [58]. P-cresol seems to negatively affect the homeostasis of colonic epithelial cells in children with ASD. When tested *in vitro*, excessive concentration of p-cresol showed deleterious metabolic and genotoxic effects on colonocytes [59]. In addition, early exposure to p-cresol may contribute to the severity of behavioral symptoms and cognitive impairment in ASD toddlers. On the other hand, propionic acid is known to have a number of direct effects on gut physiology. It increases the contraction of colonic smooth muscle, dilates colonic arteries, stimulates serotonin release, and decreases gastric motility, which could be easily related to the GI abnormalities frequently observed in many ASD patients [60]. In fact, lower plasmatic levels of propionic acid have been reported in ASD children as an aspect of metabolic alteration in gut host-microbial co-metabolism. Authors related the occurrence of lower propionic acid in the plasma to elevated levels of propionic acid in the brain [61]. Additionally, administration of neurotoxic dose of propionic acid to animals was effective in inducing autistic features. Orally dispensed propionic acid was reported to induce oxidative stress. Elevated interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interferon γ (INF-γ) confirmed the neuroinflammatory effect of propionic acid [61]. When propionic acid was injected into the cerebral ventricles of rats, the rats showed biologic, chemical, and pathologic changes that are characteristic of ASD [62]. These chemicals can also alter the intercellular spaces between the cells, resulting in the "leaky gut syndrome" that can lead to

*DOI: http://dx.doi.org/10.5772/intechopen.89375*

detrimental neurologic effects.

**spectrum disorder**

#### *Could Gut Modulation through Probiotic Supplementation Be Beneficial in Autism Spectrum… DOI: http://dx.doi.org/10.5772/intechopen.89375*

bacterial metabolites (e.g., p-cresol) and SCFA (e.g., propionic acid) is frequently described in infants with ASD [30, 49, 57]. These compounds induce intracellular signaling and modulate host gene expression related to neurotransmission systems and behavior [58]. P-cresol seems to negatively affect the homeostasis of colonic epithelial cells in children with ASD. When tested *in vitro*, excessive concentration of p-cresol showed deleterious metabolic and genotoxic effects on colonocytes [59]. In addition, early exposure to p-cresol may contribute to the severity of behavioral symptoms and cognitive impairment in ASD toddlers. On the other hand, propionic acid is known to have a number of direct effects on gut physiology. It increases the contraction of colonic smooth muscle, dilates colonic arteries, stimulates serotonin release, and decreases gastric motility, which could be easily related to the GI abnormalities frequently observed in many ASD patients [60]. In fact, lower plasmatic levels of propionic acid have been reported in ASD children as an aspect of metabolic alteration in gut host-microbial co-metabolism. Authors related the occurrence of lower propionic acid in the plasma to elevated levels of propionic acid in the brain [61]. Additionally, administration of neurotoxic dose of propionic acid to animals was effective in inducing autistic features. Orally dispensed propionic acid was reported to induce oxidative stress. Elevated interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and interferon γ (INF-γ) confirmed the neuroinflammatory effect of propionic acid [61]. When propionic acid was injected into the cerebral ventricles of rats, the rats showed biologic, chemical, and pathologic changes that are characteristic of ASD [62]. These chemicals can also alter the intercellular spaces between the cells, resulting in the "leaky gut syndrome" that can lead to detrimental neurologic effects.

Dysbiosis can also affect the functional intestinal barrier that can lead to an alteration in the intestinal permeability referred to as "leaky gut" state, a fundamental factor underlying the relationships between ASD and the GM [63]. Indeed, several reports show increased gut permeability in ASD patients [64]. Disrupted barrier function facilitates the translocation of bacterial metabolites from the gut into the bloodstream to possibly reach the otherwise sterile CNS inducing directly inflammatory reactions. One important bacterial component is the lipopolysaccharide (LPS) that was shown to increase the activity of areas deputed to the emotionalism control such as amygdale [65]. It also leads to the production of inflammatory cytokines that critically alter the physiological brain activity, modulating the neuropeptides synthesis [66]. Moreover, it has been demonstrated that the administration of low doses of LPS in healthy subject is associated to increased pro-inflammatory cytokines and plasma norepinephrine, with higher depression rates, fatigue, and apathy [67]. Consistent with this, a study showed LPS serum levels were significantly higher in autistic patients compared to heath individuals and correlated with socialization scores in an inverse and independent manner [68].
