The Human Gut Microbiome as a Potential Factor in Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a complicated neurodevelopmental disorder characterized by decreased verbal and social interactions, limited interests and activities, and repetitive behaviors [1,2]. Along with these significant conditions, ASD regularly co-occurs with other clinical symptoms, including gastrointestinal disturbances (up to 70%), motor deficits (79%), sleep problems (50–80%), and intellectual disability (45%) [3].

Autism prevalence has risen dramatically worldwide in the last few years, reaching 1 in 132, and with a remarkable increase in occurrence in boys compared with girls [2,4]. In the United States, the prevalence of ASD rose from 1 in 150 children in 2000 to 1 in 54 in 2016 [5]. The dramatic increase in ASD reduces parental productivity and increases the financial burden on families, with central expenditures being linked with special schooling [6].

For many years, a high number of studies have been conducted worldwide focusing on the potential etiology of ASD; however, its precise etiology has not been clearly identified. Gene and chromosomal abnormalities, such as fragile X syndrome (FXS); tuberous sclerosis (TSC); and potential defects in chromosomes 2q, 7q, 15q, and 16p, are shown in 35 to 40% of ASD cases. Furthermore, the ASD rate was found to be higher in monozygotic twins than in dizygotic twins and was found to be 50-fold higher among siblings who belong to families that already have ASD children [7]. Additionally, the multigenic disorder of autism has been related to epigenetic effects [8]; nevertheless, no specific gene has been identified as being associated with all cases of ASD. Recently, 100 to 800 genes or genomic regions have been implicated in ASD etiologies [9,10].

Several studies have found that 60 to 65% of autism occurrence could be explained by prenatal, natal, and postnatal environmental risk factors (Figure 1). Prenatal risk factors involve maternal infection, maternal physical health, the health condition of pregnant women, folate and iron deficiency, and drug use in pregnancy. Natal risk factors include fetal complications, umbilical cord complications, hypoxia (lack of oxygen), cesarean delivery, abnormal presentation of the fetus, and abnormal gestational age (preterm or post-term). Postnatal risk factors include breastfeeding, air contamination, antibiotic intake, and nutrition factors [10,11,12,13,14]. Environmental risk factors can directly influence the neuronal activities of the growing brain of the fetus [13]. These environmental risk factors are largely found to shape the intestinal microbiota [15]. Therefore, the lack of an imprecise cause of the development of autism disorder has prompted scientists to look into other putative triggers, such as the intestinal microbiota.

The human gut comprises millions of microorganisms, and it has been suggested that a well-balanced gut microbial composition helps to maintain microbial homeostasis. At the same time, alterations in microbial composition frequently end with a negative influence on the health condition of human beings [16]. Presently, the gastrointestinal (GI) tract is considered a new organ that makes numerous metabolites and neuroactive substances. About 40% of all human metabolites are generated by the gut microbiome [17,18]. As a result, any imbalance in the community and quantity of gut microbiota during a critical time in a child’s development may impact the central nervous system (CNS) and enteric nervous system (ENS), which comprise the microbial gut–brain axis [18]. This axis describes how the gut flora can communicate with the brain and how they can impact each other [14].

Various emerging findings have revealed an alteration in the gut microbial composition in ASD individuals compared to neurotypically developing children [19]. Interestingly, GI symptoms, including abdominal pain, gastroesophageal reflux, flatulence, and constipation, have frequently been described to occur at rates of 9–84% in ASD children [20]. This avenue of analysis is essential for defining the role of microbiota dysbiosis in ASD and launching a possible treatment for ASD patients. Therefore, this paper aims to review the role of gut microbiota dysbiosis in the pathology of ASD, focusing on the microbiota–gut–brain axis. Moreover, the current review examines the present therapeutic approaches for ASD. Therefore, the review adds to our understanding of the responsibility of gut microbes in influencing ASD in humans.

The methods of this review article were based on the utilization of virtual databases, including PubMed and Science Direct, to search for all related published studies, whereas the statistics of the prevalence of ASD were taken from the website of Centers for Disease Control and Prevention (CDC). All studies included in this review were published from 2003 to 2021. The selection was based on the keywords “ASD“, “autism”, “gut” “microbiome”, “microbiota”, “gut-brain axis” “probiotics”, and “fecal transplantation”. Nearly 235 articles were found, and those examining the gut microbiota and general neurodegenerative disorders—particularly autism spectrum disorder—were included in this review article.

Human beings have co-evolved with a massive number of microorganisms that colonize almost every part of the body, particularly the skin, eyes, respiratory pathway, urogenital pathway, and intestine [21]. These microorganisms include bacteria, fungi, viruses, archaea, and protozoa [22]. The community of these microbes is called the microbiota, where the term microbiome indicates the genomes of these microorganisms [23]. It is believed that a series of microbial establishment events in the gut starts during the prenatal period, as proposed by the existence of microbes in the placenta, amniotic fluid, meconium, and the blood of the umbilical cord [24]. Interestingly, the significant periods of alteration in the evolving microbiota overlap partly with the timespan for the development of the brain [25]. The colonization of the newborn baby gut begins during birth—e.g., the newborn infant born through the vagina becomes covered with the mother’s vaginal microbes, or of the mother’s skin in the situation of a cesarean delivery (C-section) [26]. About 75% of the feces microbiota of vaginally born babies were found to be related to their mothers’ fecal microbiota, whereas, in C-section babies, this percentage decreases to ~41% [27].

Following delivery, the baby is introduced to bacteria during breastfeeding, through the intake of food, and from the surrounding environment [28]. Many research studies have found that the diversity of the gut microbiota is decreased in formula-fed compared with breastfed children [29]. Key alterations in the gut microbial composition occur throughout the weaning period because the infant moves from consuming formula or breast milk to solid food [30]. In adolescence, microbial diversity and functional capacities develop toward an adult-like microbial profile, with each individual having a unique microbial community [14,31,32]. No two people have the exact same microbial community—not even monozygotic twins [33]. In adulthood, nutrition and antibiotics are the essential aspects that impact the composition of the gut microbiota across the human lifespan [25]. Consequently, the description of the adult microbiota state as ‘stable’ is somewhat imprecise, as the gut microbial community alters over time and can be restored after changes [34] (Figure 2).

Metagenomic analysis has revealed that the gut of the human body encodes about 3.3 million gut microbial composition genes, which is 150-fold higher than that of the whole human genome. Bacteria represent the most prevalent members of the intestinal microbial community, since the human body contains 1013 human bacterial cells, with a human-to-bacterial cell ratio of approximately 1:1 [36].

Several analysis approaches have recently enabled scientists to detect and quantify gut microbial components by examining nucleic acids (DNA and RNA) collected from feces. Most of these methods rely on DNA extraction and the amplification of the 16S ribosomal RNA gene (rRNA) [37]. 16S ribosomal RNA (rRNA) sequencing provides a taxonomic characterization of microbial communities. Whole-genome shotgun (WGS) (whole DNA sequencing) is able to derive the highest-quality data of functional and organismal human microbial communities [38]. Based on the available data from the Human Microbiome Project and Metagenomics of the Human Intestinal Tract (MetaHIT), about 2776 microorganisms have been extracted from human feces and categorized into 12 bacterial phyla, with Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria accounting for up to 90% of total bacteria, while Verrucomicrobia and Fusobacteria phyla exist at a low abundance [21].

Since mammals cannot synthesize many vital nutrients, the gut microbes perform essential functions for the host, delivering necessary nutrients by digesting dietary compounds, protecting against opportunistic microbes, and contributing to the integrity of intestinal epithelial barriers [19]. Based on all these features, the complex human–microbiota interconnection can be considered an existent superorganism whose disturbance could lead to disease onset [39]. As a result, this path may help us to develop beneficial microbiota-associated therapies in the upcoming years.

There are many reasons why researchers link ASD symptom development with the gut microbial composition. For example, in 2019, Sharon and colleagues reported that germ-free (GF) mice display ASD-like behavior after being colonized with fecal microbiota from children with ASD. In this study, gut microbiota were transplanted to GF mice from human donors with autism, or from their typically developing (TD) siblings as a control. Mice colonized with ASD microbiota displayed more autistic behaviors compared to the TD mice. The team also found that the ASD group had a different abundance of Clostridiaceae, Lactobacillales, Enterobacteriaceae, and Bacteroides in comparison with the TD group. They also observed that mice that were treated with gut microbiota from ASD patients displayed an alternative splicing of many ASD-related genes in the brain. Moreover, some metabolite profiles were detected to be decreased in the ASD group—particularly 5-aminovaleric acid (5AV) and taurine [40] (Table 1).

On other hand, compared to neurotypical children, ASD patients were largely found to have altered gut microbial compositions [41]. Nonetheless, no particular microbial species have been found to be consistently changed in all ASD microbial studies, which could be related to changes in different aspects such as diet, age, gender, population, and autism severity [16,42]. Although the alterations in gut microbial in ASD patients are not always consistent between studies, ASD patients frequently have microbial imbalances of many types, most notably a decreased Bacteroidetes/Firmicutes phyla ratio, which could be the result of a decrease in the relative abundance of Bacteroidetes [36]. The Bacteroidetes phylum is responsible for polysaccharide digestion. Therefore, this research could support the theory that ASD patients have an abnormal digestion of carbohydrates and mucosal dysbiosis in the gut [1,43]. In other studies, a high level of Actinobacteria phylum was noticed in autistic patients in comparison with the control group. ASD patients were also found to have a high abondance of the Betaproteobacteria class and high levels of Lactobacillus, Ruminococcus, and Escherichia-Shigella species. In contrast, the prevalence of Bifidobacterium and Enterococcus species was decreased [42,44,45]. Kang and others revealed a decline in the abundance of the genera Coprococcus, Prevotella, and unclassified Veillonellaceae in ASD patients [46]. In a previous study, certain Prevotella species (P. ruminicola, P. oralis, and P. tanneries) were shown to be decreased in irritable bowel syndrome (IBS) patients [36]. This study leads to the theory that ASD-related gastrointestinal symptoms are linked to a change in the microbial balance [46]. In other previous studies, the abundance of the genus level of Akkermansia was found to be low in ASD children, with the level of Desulfovibrio spp. being increased [44,47]. The latter bacteria are considered to be harmful, as they may exacerbate autistic behaviors and gastrointestinal problems [48]. In addition, although Sutterella spp. is usually uncommon in healthy microbiota compositions, an elevated abundance was found in the caecum and ileum intestinal tissues of ASD children [26]. Compared to neurotypical healthy individuals, ASD children were shown to have a significantly increased abundance of Clostridium cluster groups XVIII and Clostridium bolteae [42,43]. In addition, Alshammari and others observed a significantly higher rate of Clostridium perfringens in ASD children than in the controls [43,49]. Clostridium produces neurotoxins that exacerbate autistic symptoms and has been linked to the Childhood Autism Rating Scale (CARs), which determines the severity of ASD [41,50,51]. In addition, C. perfringens can produce toxins, especially the Beta2 toxin, which is associated with an increased ratio of GI abnormalities such as food poisoning and diarrhea [36]. Fisher and others found that the feces samples of ASD patients were 79% beta2 toxin, whereas they were only 38% in the control group [36].

In addition to bacterial changes, ASD patients have been found to have gut fungal dysbiosis. For example, Candida albicans, which produces ammonia and other toxins that are likely associated with autism-related behavior, was found in the guts of autistic children more frequently than in those of non-ASD children [51,52]. Furthermore, a significant increase in Saccharomyces cerevisiae was discovered in ASD individuals compared to non-autistic individuals. On the other hand, Aspergillus versicolor was found significantly less often in ASD patients. These alterations indicate a possible role of immune pathways in triggering ASD. S. cerevisiae can regulate immune function by activating TLR ligands and increasing TNF-α and IL-6 production, while A. versicolor has the potential to produce anti-inflammatory metabolites [53].

Although these studies clearly show modifications in the composition of the gut microbiota in ASD children, some research suggests that the variations between neurotypical and ASD children’s microbial composition could be due to the overuse of antibiotics by autistic children. Antibiotics influence gut homeostasis by targeting pathogens and commensal bacteria [14,28]. This may illustrate why the gut microbiota of children under three years old who have been given antibiotics is less diverse [14,28]. Additionally, the use of antibiotics by pregnant women is also related to a high risk of autism occurrence [14]. On the other hand, oral vancomycin treatment, a well-known antibiotic effective against clostridia, resulted in a considerable improvement in the symptoms of patients with ASD [36].

This area of research is still growing, so more studies with large sample sizes of autistic and neurotypical children who are not treated with antibiotics are needed in order to detect the specific function of gut microbiota dysbiosis in the onset of autism spectrum disorder.

The gut–brain axis refers to a bidirectional connection between the gut and the brain. It can also extended to involve the microbiota as an essential part of this triangle dialogue [21]. This bidirectional pathway consists of both efferent and afferent signals. Afferent signals transmit from the gastrointestinal tract to the brain and involve the enteroendocrine system, cytokines, metabolites, gut products, and neuroactive molecules. Efferent signals start from the brain to the gut wall and include neuroendocrine and autonomic regulation [20,62]. In this pathway, 90% of vagal fibers between the brain and gut are afferent, suggesting that the intestine is more of a transmitter than a receiver [63,64,65]. This bidirectional link comprises one or more of the following avenues (Figure 3).

In the last few decades, the rapid rise in the rate of ASD has demonstrated that autism cannot be caused only by genetics. Therefore, scientists have examined the relationships between genetics and the environment, especially studying the role of epigenetics in causing ASD [8]. Epigenetics investigates the ways in which environmental and lifestyle factors influence DNA expression without changing the DNA sequence, which can be transmitted from one generation to another via germline cells. These epigenetic modifications can control when, or even if, a specific gene turns on and off in a cell or organism [112,113].

DNA methylation, post-transcriptional histone modifications, and gene expression regulation by non-coding RNAs are some examples of epigenetic regulation [114]. DNA methylation has been related to the etiology of nervous disorders, including ASD [115]. For example, a methylome analysis study of the human placenta exhibited a significantly higher level of a methyl group in patients with ASD through the use of pyrosequencing [116].

Several compelling pieces of evidence suggesting that the gut microbial community is directly responsible for initiating epigenetic modifications [117]. Exchange talk between microbic metabolites and external effectors such as antibiotics, nutrition, and other environmental factors can shape the epigenome (temperature, oxygen, and pH) [118]. Commensal bacteria in the gut can synthesize folate, vitamin B12, and choline, all of which are fundamental in the production of a methyl group donor (6-methyltetrahydrofolate) and the formation of S-adenosylmethionine (SAM), which is the main methyl donor in the DNA methylation process [119]. For example, Bifidobacteria and Lactobacillus species are known for folate synthesis [120]. Another critical microbial metabolite that affects epigenetics is butyric acid, a potent inhibitor of histone deacetylases [121], which removes the acetyl group from histone proteins, letting the proteins re-associate with DNA and preventing DNA transcription. Moreover, the latest suggestion shows that some endosymbiotic bacteria make small non-coding RNAs that influence host processes [122].

Based on the above-mentioned findings regarding the involvement of epigenetics in ASD, one can assume that dysbiosis in the gut microbiota composition, particularly in the early periods of development, could directly switch a specific gene on or off. In this situation, the excessive use of antibiotics may affect microbial diversity and turn on a particular gene related to autism.

There is no current reliable therapy for treating patients with ASD. However, because of the increasing amount of data regarding the role of gut microbial dysbiosis in ASD, researchers are currently focusing on strategies for treating such a disease by modulating the gut microbial community as a potential therapeutic approach. This approach involves oral prebiotic, probiotic, dietary, and/or fecal microbiota transplantation (FMT) as well as microbiota transfer therapy (MTT; Figure 4 and Table 2).

Mounting evidence from human and animal studies suggests that gut microbial-targeting therapy may be beneficial as a new and safe method for treating ASD patients. Antibiotics have been used as a possible treatment for ASD patients, but antibiotics influence gut homeostasis by targeting pathogens and commensal bacteria. Thus, antibiotics are not a possible option for long-term therapy for ASD. Probiotics can colonize the gut and restore the composition of bacterial populations, which, in turn, has been found to reduce autism-related symptoms. Though probiotics are commonly safe to use, a study by Rondanelli et al. [131] advises that individuals with serious underlying medical illnesses or weakened immune systems should not take probiotics, since some individuals with these circumstances were found to have bacterial or fungal infections as a consequence of probiotic intake [131]. Prebiotics serve as food for commensal bacteria, so they stimulate an increase in beneficial bacteria that are found naturally in the body and improve digestive health. Studies on ASD patients using prebiotics are limited, and there is a lack of available solid data [88]. Multiple studies have shown that ketogenic diets (KD), gluten-free and casein-free (GFCF) diets, and supplementation with omega-3-fatty acids have beneficial effects on the health of children with ASD, but the evidence available is limited and weak. Marí-Bauset et al. found some possible side effects of the GFCF diet, such as calcium deficiency and a lack of essential amino acids, resulting in decreased bone density and frequent bone fractures. Moreover, ASD patients who followed the GFCF diet needed more supplementation with vitamin D [123]. Fecal microbiota transplantation (FMT) can modify the gut microbiota composition by transplanting fecal microbiota from healthy donors to ASD patients [132]. FMT has emerged as a safe and promising therapeutic approach and can restore metabolites and immune function. However, FMT could have future unexpected health effects, since there are many microbes in the gut that have not been determined yet which may introduce pathogenic bacteria into the host’s intestinal system. Therefore, to obtain the most benefit from fecal transplantation, further strict donor screening is needed to minimize the risk of FMT. Microbiota transfer therapy (MTT) was found to reduce gut and ASD-like symptoms and regulate the gut microbiota of autistic individuals [124].

The increased rate of ASD shows an urgent need to detect the etiology and pathogenesis of autism. In the last few decades, accumulating evidence has implicated gut microbial dysbiosis in the etiology of ASD, as it has an essential role in various important body functions involving the development of the central nervous system (CNS) and neuropsychological homeostasis, in addition to the health of the gastrointestinal (GI) tract. There are several pathways by which the microbiota of the gut or their components affect the brain. A deeper understanding of these pathways could open up novel avenues that allow the beneficial treatment of ASD patients, reducing ASD-related symptoms and improving patients’ quality of life.

Even though gut microbial dysbiosis has been linked to ASD pathogenesis, at present, it is not likely to define a single microbe as a hallmark of ASD. This is due to the lack of consistent analysis approaches as well as the heterogeneity of enrolled participants—including participants’ age and sex, the different scales used for the evaluation of ASD symptoms, the presence/absence of gastrointestinal symptoms, and the different dietary lifestyles followed. Moreover, most of the reports enrolled a small number of ASD individuals who do not represent most of the ASD population. However, in future studies of FMT, the use of large sample sizes may lead to the identification of definite combinations of beneficial microbes that can be used to cure ASD.

To deeply examine the role of intestinal microbes in ASD, additional studies should focus on another unexplored area that can help identify the distinctive ASD microbiome. For example, current studies have mostly explained changes in gut bacteria—only a few studies have focused on fungi, and no studies have been carried out on other gut microbiota, such as protozoa, viruses, and archaea. Likewise, the application of multi-omics approaches in future research is highly recommended in order to gain more conclusive outcomes.

Writing—original draft preparation: A.A.; review and editing: S.A., N.A., A.B. All authors have read and agreed to the published version of the manuscript.

This research received no external funding.

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No new data were created or analyzed in this study. Data sharing is not applicable to this article.

The authors declare no conflict of interest.