Increasing evidence suggests the balance and diversity within the bacterial population are essential in maintaining proper function of the gastrointestinal tract and immune system as well as human homeostasis. On the other hand, there are a wide range of indicators that propose an imbalance of the gut microbial ecosystem may lead to inflammation and immune activation in several disorders such as gastrointestinal diseases, cardiovascular disease, metabolic and psychiatric disorders, allergy, or asthma 6,12,15,16,41.
The pathogenesis of ASD is complex, and apart from genetic factors, environmental, factors such as the intestinal community, may play a key role in the symptomology of ASD. The composition of gut microorganisms that increase susceptibility to autism development, as well as evidence linking autism symptoms and intestinal dysbiosis, have yet to be fully explained 19–21,25,43. However, frequent occurrence of GI symptoms in ASD children suggest the involvement of the gut microbiota in gastrointestinal pathophysiology which then constitute potential diagnostic and therapeutic targets. It was suggested that dietary intervention (gluten-, casein-, and soy-free diet), probiotic/prebiotic treatment, microbiota transfer therapy, or targeted antibiotic therapy could be a new strategy for treatment. It could help children with chronic gastrointestinal disorders and may reduce ASD symptoms by improving language, cognitive skills, and behavioral deficits (Doenyas 2018; Fattorusso, Di Genova, Dell'isola, et al. 2019; Kang et al. 2019; Ng et al. 2019).
Recent studies examining the association of microbiota and children with autism suggest excessive use of antibiotics in ASD individuals may cause an overgrowth of certain Clostridum species such as C. tetani, C. perfringens, or C. difficile. According to the hypothesis linking the occurrence of Clostridium with the etiopathogenesis of autism, an overgrowth of some toxin-producing Clostridum species can expose ASD children to high levels of microbial neurotoxic metabolites. This thereby affects normal nervous system development and leads to the exacerbation of gastrointestinal problems (Argou-Cardozo and Zeidán-Chuliá 2018; Ding et al. 2017; Fattorusso, Di Genova, Dell'Isola, et al. 2019; Finegold et al. 2017; Strati et al. 2017).
Moreover, the anaerobic bacteria Clostridium and Bacteroides are sources of short-chain fatty acids (SCFA), such as propionic, acetic, butyric, and valeric acid, usually produced during fiber fermentation. These metabolites are believed to be involved in gut immune system function, modulation of the nervous system through the gut-brain axis, and host cell gene expression 26,34,51. SCFAs can induce widespread effects on the human organism, but an imbalance in their levels may change intestinal homeostasis and cause peripheral inflammation. SCFAs reach the brain through blood circulation and affect its development by modulating production of serotonin and dopamine. High concentrations of propionic acid, a significant neurotoxic metabolite, may disrupt brain function, resulting in developmental delay or regression 26,52−54.
However, the presence of specific Clostridium species, clusters, and their content in the intestinal microbiome of ASD children is still under discussion. Moreover, current results are often inconclusive, and the contribution of selected species in ASD etiology have yet to be fully explained. Our analysis showed no significant differences in the levels of Clostridium spp. within the groups. The results, therefore, are similar to those of Wang and Iovene 38,55 but are not consistent with other reports that found increased Clostridium in the stool of ASD children34,50,56,57.
Bacterioides spp. and Clostridum spp. are defined as bacteria associated with fiber fermentation and SCFA/propionic acid production. It has been suggested that neurodevelopmental disorders in ASD patients correlate with impaired propionic acid metabolism and changes in propionate producing bacteria 54,58. In our analysis, the level of Bacteroides is unchanged in all analyzed groups. These findings are comparable with those of Parracho et al. and Ma et al. 56,59 but contrasts other studies where increased Bacteroides in ASD patients has been reported 34,51. Moreover, in the microbiome of the ASD group, a significant increase of Proteobacteria phylum, particularly species belonging to Enterobacteriaceae, was observed 34,51,60. However, our analysis showed no significant changes in the abundance of this family in stool samples of any studied groups, except a higher level of Klebsiella species in the healthy group. This result is compatible with Adams' observation 29.
Additionally, in healthy controls, a significantly positive and strong correlation of Escherichia coli and Enterococcus spp. was noted. It is well-known that certain strains of E. coli and Enterococcus have probiotic properties and can activate the gut mucosal immune system by increasing antibody quantities and cytokine production and also improve the barrier function of the intestinal epithelium 61–63. Cukrowska et al. has reported the presence of probiotic E. coli Nissle 1917 in infant's intestines may enhance the humoral immune system response, especially the induction of specific IgA and IgM antibodies 64. Hafez has demonstrated that this beneficial strain may regulate mucin gene expression, thereby altering the intestinal mucus layer and indirectly regulating the gut immune system 65.
Some studies have also indicated varying levels of probiotic bacteria such as Lactobacillus and Bifidobacterium in the intestines of ASD and neurotypical subjects 27,29,30,34,38,51. Our results showed lower levels of Bifidobacterium in ASD group, which is compatible with several other studies 29,34,38,51. We speculate this may be due to a derangement of the probiotic bacteria population in the intestines of autistic children. On the other hand, similar numbers of Bifidobacterium in ALG and healthy groups may be a compensatory mechanism. Allergies are a chronic inflammatory diseases, and this group of bacteria shows strong anti-inflammatory properties. Recently published studies have reported that Bifidobacterium strains may inhibit the inflammatory response and exert an immunomodulatory effect by stimulating IL-10 or IL-12 synthesis by dendritic cells 66,67. Furthermore, the presence of both probiotic bacteria in the intestines contributes to maintenance of the epithelial barrier integrity and protects against an overgrowth of pathogens 55,68. Additionally, they can impact the metabolism of toxins, drugs, and some dietary compounds as well as gut epithelial cell proliferation 68–70. Interestingly, both genera may produce γ-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the brain 26,69. According to some studies, lower GABA levels are correlated with anxiety and social disorders in ASD individuals 71,72.
Moreover, some Lactobacillus and Bifidobacterium strains are the main components of probiotic supplements. Growing clinical evidence suggests the consumption of oral probiotics reduce GI discomfort, modulate the stress response, and improve mood and anxiety symptoms in patients with ASD 29,30,73,74. However, in our analysis, we observed a strong negative correlation between probiotic bacteria and Akkermansia muciniphila and Faecalibacterium prausnitzii levels in ALG and ASD groups using probiotics. We assume this may be due to the dominant role of some probiotic strains or as a result of nutrient competition. Both A. muciniphila and F. prausnitzii are considered biomarkers of healthy intestinal flora and modulators of immune system 75,76. Additionally, Faecalibacterium may regulate the expression of interferon-gamma (IFNγ), which plays an indirect role in neuroplasticity and synapse formation 26,77. Based on these factors, it can be assumed in children with these associated diseases, selection of appropriate probiotic strains is important, and probiotic therapy should be performed on the basis of previous microbiota analysis.
In our studies, we estimated the content of fecal fungi, especially Candida genus, in ASD children. The healthy gut is colonized by yeast and good bacteria living in balance with one other. Most Candida species are harmless commensals, but when intestinal homeostasis is disturbed, they can cause infections called candidiasis. Yeast infections have been rarely investigated in autistic individuals. Our studies have shown no significant differences between groups. However, some investigators report substantial growth of Candida, particularly Candida albicans, in ASD patients 27,55,78,79, Contrary to these results, Adams et al. did not confirm these findings 29. The potential role of the Candida species in ASD etiology is unclear, and further studies are needed. It is believed that an overgrowth of Candida spp. may induce autistic behavior through excessive production of ammonia which then is converted to beta-alanine, a non-essential amino acid structurally similar to the inhibitory neurotransmitter GABA 79,80. Additionally, a high abundance of yeast may impair the absorption of both carbohydrates and mineral elements, as well as affect the release and accumulation of toxins 78,80. Moreover, enormous growth of Candida in the gut of autistic individuals may aggravate GI abnormalities by dysregulation of cytokine release 27.