5.1. Changes in Microbial Composition in Autistic Children
A regularly observed phenomenon in the faeces of autistic children is a significantly decreased ratio between the phyla Bacteroidetes to Firmicutes [
49,
53,
76,
87], which pointed to elevated numbers of Firmicutes in contrast to decreased levels of Bacteroidetes. Conflicting data were reported by De Angelis et al., who found in a study with thirty subjects that Firmicutes counts were lower than Bacteroidetes in autistic children when compared to healthy controls [
40]. These results, however, were not statistically significant [
40]. Moreover, the phyla Fusobacteria and Verrucomicrobia were also represented in lower concentrations (
Figure 3) in the faeces of ten autistic children compared to ten healthy controls [
40]. Significantly elevated bacteria in autistic children were
Akkermansia muciniphila,
Anaerofilum,
Barnesiella intestinihominis,
Clostridium spp,
Dorea spp, the family Enterobacteriaceae,
Faecalibacterium spp (especially
Faecalibacterium prausnitzii),
Roseburia spp,
Parasutterella excrementihominis,
Prevotella copri,
Prevotella oris and
Turicibacter spp.
Escherichia coli was significantly decreased in autistic children [
40]. Other higher represented spp were
Aeromonas,
Odirobacter splanchnicus,
Parabacteroides,
Porphyromonas,
Pseudomonas, and
Turicibacter sanguinis. Significantly decreased bacteria in autistic children were
Bifidobacterium,
Fusobacterium,
Oscillospira,
Sporobacter,
Streptococcus and
Subdoligranulum. Lower represented genera in autistic children were
Collinsella spp except
Collinsella aerofaciens,
Enterococcus spp,
Lactobacillus,
Lactococcus and
Staphylococcus [
40]. Kang et al. compared the intestinal flora of twenty autistic children with GI problems to the intestinal flora of twenty neurotypical children. They found that the significant lower bacterial diversity found in autistic children [
20,
21] correlates with the severity of GI symptoms. No major differences were detected at phylum levels; however, this discrepancy could be a result of the parents collecting and freezing the stool samples and not the research team. Non-autistic children had a significantly higher abundance of the genus
Coprococcus and class Prevotellaceae compared to autistic faecal samples.
Prevotella spp are commensal gut microbes, specialised in degrading plant polysaccharides and synthesising vitamin B1. Lower abundance of
Prevotella spp could therefore result in a vitamin B1 deficiency. In addition, Veillonellaceae were found in lower abundance in autistic children. Small differences were observed in
Sutterella genus, being less abundant in autistic children [
20]. These data reveal a change in microbiota composition in autistic children that may have biochemical and functional consequences on the host.
Intestinal biopsies from the caecum and the terminal ileum of twenty-three ASD children showed significantly increased numbers of
Sutterella spp compared to nine healthy controls.
Sutterella spp are normally scarce in a healthy microbiota and were found in none of the controls, but in twelve of the twenty-three ASD patients [
88]. The microbial composition was also studied in duodenal biopsies comparing nineteen autistic children with twenty-one healthy controls [
89]. The study did not find any major differences between the phyla, but some alterations were observed at genus und species levels. The genus
Burkholderia was significantly increased in autistic participants compared to controls. The genera
Actinomyces,
Oscillospira,
Peptostreptococcus and
Ralstonia were elevated and the genus
Neisseria was significantly under-represented in autistic children compared to controls. The genera
Bacteroides,
Devosia,
Prevotella and
Streptococcus were also decreased. Also, lower abundances of
Escherichia coli were observed [
89]. Kushak et al. could not find major differences at phylum level as above-mentioned studies [
40,
49,
53,
76,
87]. This could be due to the fact, that all the other studies investigated microbial composition from stool samples and not biopsies from duodenal mucosa. The stool samples represent, by and large, microbial composition of the large intestine [
90]. Kushak et al., on the other hand, represented microbial composition of the small intestine. Kushak’s results suggested that microbial changes in the large intestine could have a greater impact on the pathology of ASD than the changes in the small intestine. Therefore, a better representation of the microbial gut milieu could be achieved by collecting mucosal samples from all over the GI tract for comparison of the microbiota resulting in a better understanding of the inter-relationships of the microbiota functioning and the interaction with the host.
Biopsies from the ileum and the caecum of fifteen ASD children (seven controls) showed a lower abundance of Bacteroides leading to a significantly higher Firmicutes to Bacteroidetes ratio [
53]. Levels of Clostridiales (Firmicutes) were slightly elevated in ASD group, especially the families Lachnospiraceae and Ruminococcaceae and the genus
Faecalibacterium. The class Betaproteobacteria (Proteobacteria) was significantly higher abundant in faecal samples of the caecum in ASD patients, within the class Betaproteobacteria the family Alcaligenaceae was the highest abundant [
53]. Concomitant with increasing levels of Bacteroidetes the mRNA levels of SGLT1 in ileum and caecum significantly increased, whereas the mRNA levels of sucrase isomaltase (disaccharidase) decreased in the caecum. With increasing mRNA levels of sucrose isomaltase a significant decreased amount of Firmicutes was observed in the caecum [
53]. These associations may be of therapeutic interest, as they demonstrated how bacterial composition influenced expressions of the host’s sugar transporters and enzymes to consequently alter their nutrients’ availability. Therefore, directing the bioactive therapeutic molecules to the small intestine or the colon may be a practical approach to circumvent systematic absorption achieving a specific modulation of the microbiota in these organs.
Sibling control studies delivered discordant results. A Slovakian study enrolling twenty-nine participants including autistic children, their siblings and healthy children found that the amount of
Lactobacillus spp was significantly increased (
Figure 3) in the faeces of autistic children compared to siblings and healthy controls. Amount of
Desulfovibrio and
Clostridia spp were also increased, whereas
Bifidobacterium spp were decreased in autistic children (
Figure 3) [
49]. The authors associated the severity of behavioural symptoms in social interaction, communication and restricted/repetitive behaviour in ASD with the amount of
Desulfovibrio spp present. Therefore,
Desulfovibrio spp were considered pathogenic microbes. Interestingly, non-autistic siblings of autistic children showed lower abundance of Bacteroidetes and higher abundance of Firmicutes compared to healthy controls with no family history of ASD [
49]. Furthermore, non-autistic siblings had lower abundance of
Clostridia and
Desulfovibrio spp and significantly lower counts of
Bifidobacterium spp compared to autistic children. Therefore, it was postulated that the levels of bacterial spp could be the tipping factor between autistic or healthy phenotypes [
49]. In contrast, Gondalia et al. found no significant difference in microbial composition after analysing fifty-one autistic children and their fifty-three non-autistic siblings [
91]. Similarly, a different study including fifty-nine autistic children and forty-four non-autistic siblings also found no significant difference in the composition of the gut microbiota [
92]. These data suggest that the microbiota of siblings are similar and independent from their autistic phenotype [
91], probably as a result of the shared environment and genetic makeup [
93].
Higher occurrence of
Clostridium spp in the gut was associated with disease severity using the Childhood Autism Rating score (CARs score) [
15]. Significantly higher counts of the bacterium
Clostridium perfringens were found in faecal samples of thirty-three autistic children compared to thirteen controls. Especially the spp
Clostridium perfringens producing the beta2-toxin gene was significantly elevated in autistic children. The authors also found a significant relationship between the abundance of the beta2-toxin gene and occurrence of ASD [
21]. Herbicides were considered to preserve
Clostridia spp and harm beneficial bacteria [
94]. A review suggested that a sub-acute tetanus infection with a
Clostridium spp might be the cause of some cases of ASD. Infection with the pathogen
Clostridium tetani only occurs in dysbiotic GI tracts, as it is an opportunistic pathogen. It may be inactive for several months until favourable conditions allow its growth.
Clostridium tetani produces tetanus neurotoxin, which crosses the intestinal barrier. Tetanus neurotoxin is then transported via the vagal nerve to the nucleus solitarius and subsequently to the whole CNS. Tetanus neurotoxin inhibits the release of synaptic vesicles containing neurotransmitters by irreversibly cleaving synaptobrevin, a membrane-associated protein, which is important for vesicle stability. Synapses with cleaved synaptobrevin degenerate and lower synaptic activity correlates with diminished social behaviour found in ASD (
Figure 4). Tetanus neurotoxin targets inhibitory neurons releasing GABA or glycine. Purkinje and granular cells in the cerebellum express receptors for the neurotoxin and the purkinje cells release GABA as neurotransmitter, making them vulnerable to the toxin. This is in line with the observation of decreased Purkinje and granular cells in autopsies of autistic children [
43].
The yeast Candida appears to play a role in autistic children as well. Strati et al. found significantly elevated abundances of the bacterial genera
Collinsella,
Corynebacterium,
Dorea and
Lactobacillus in the faeces of forty autistic children when compared to healthy children. Lowered levels of
Alistipes,
Bilophila,
Dialister,
Parabacteroides and
Veillonella were also found in autistic children. Additionally, the authors found the yeast
Candida to be present at increased rates in autistic children. Constipation could be associated with higher levels of
Escherichia,
Shigella and
Clostridium cluster XVIII and lower levels of
Gemmiger and
Ruminococcus [
87].
Lactobacillus spp stimulate the immune system to produce IL-22. IL-17 and IL-22 together inhibited the overgrowth of
Candida spp, however in autistic population, the altered diversity of microbial community favoured the growth of
Candida spp. Furthermore,
Candida spp, once established in the gut, prevented recolonization of commensal microbes [
87]. Several studies found notably higher abundance of
Candida spp, especially
Candida albicans in the faecal samples of autistic children compared to healthy counterparts [
15,
95]. Kantarcioglu et al. investigated the abundance of
Candida spp in the gut of 415 autistic children (and 403 controls), which was elevated. Reportedly this yeast is associated with some autistic behaviour and 60 % of the healthy population is estimated to be asymptomatic carrier of
Candida spp [
95]. Normally,
Candida cannot grow in the healthy microbial environment due to the competition for space and nutrients and suppression by commensal bacteria. However, in a dysbiotic environment as frequently observed in the autistic population, the yeast proliferates and produces ammonia and toxins, which were reported to increase autistic behaviour [
95].
Candida spp also cause malabsorption of minerals and carbohydrates potentially playing a role in the ASD pathophysiology.
A simulation by Weston et al. showed the interdependency between the anti-inflammatory genera
Bifidobacterium and the pro-inflammatory
Clostridia and
Desulfovibrio.
Bifidobacterium is inhibited by lysozyme and the growth of
Desulfovibrio. To some extent,
Desulfovibrio thrives on metabolites produced by
Bifidobacterium. Growth of
Clostridia is inhibited by lysozymes and by a higher abundance of
Bifidobacterium. The authors claimed that the growth of
Clostridia in the gut with low abundances of Bifidobacterium is a key risk for the development of ASD [
96]. Another simulated study analysed the microbiome coding for enzymes involved in the metabolism of glutamate and found that these enzymes were underrepresented in autistic microbiome compared to healthy ones. Glutamate is a constituent of the important peptide glutathione, which is an antioxidant and therefore reduces oxidative stress in the cell. This amino acid is also an excitatory neurotransmitter. An imbalance in the CNS between excitation and inhibition has been postulated to contribute to ASD [
97]. A general observed trend is that
Bifidobacterium spp are scarcer represented in the guts of autistic children, whereas
Clostridia spp are higher abundant [
98].
Detecting altered microbial composition is especially important to understand how microbial metabolites can modulate gut and neuronal functions. Future studies are needed to establish a possible causation. Nevertheless, a significantly changed microbial composition was found in the GI tracts of autistic population pointing to a correlation between the microbiota and the occurrence of ASD. However, some of the mentioned results contradict each other. Conflicting results have been found for example for Oscillospira spp [
40,
89], Collinsella spp [
40,
87] and Parabacteroides spp [
40,
87]. Therefore, conclusive evidence is yet to be established by future studies.