This study aimed at investigating the fecal microbiota and metabolome of children with Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) and autism (AD) in comparison to healthy children (HC). Bacterial tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) of the 16S rDNA and 16S rRNA analyses were carried out to determine total bacteria (16S rDNA) and metabolically active bacteria (16S rRNA), respectively. The main bacterial phyla (Firmicutes, Bacteroidetes, Fusobacteria and Verrucomicrobia) significantly (P<0.05) changed among the three groups of children. As estimated by rarefaction, Chao and Shannon diversity index, the highest microbial diversity was found in AD children. Based on 16S-rRNA and culture-dependent data, Faecalibacterium and Ruminococcus were present at the highest level in fecal samples of PDD-NOS and HC children. Caloramator, Sarcina and Clostridium genera were the highest in AD children. Compared to HC, the composition of Lachnospiraceae family also differed in PDD-NOS and, especially, AD children. Except for Eubacterium siraeum, the lowest level of Eubacteriaceae was found on fecal samples of AD children. The level of Bacteroidetes genera and some Alistipes and Akkermansia species were almost the highest in PDD-NOS or AD children as well as almost all the identified Sutterellaceae and Enterobacteriaceae were the highest in AD. Compared to HC children, Bifidobacterium species decreased in AD. As shown by Canonical Discriminant Analysis of Principal Coordinates, the levels of free amino acids and volatile organic compounds of fecal samples were markedly affected in PDD-NOS and, especially, AD children. If the gut microbiota differences among AD and PDD-NOS and HC children are one of the concomitant causes or the consequence of autism, they may have implications regarding specific diagnostic test, and/or for treatment and prevention.

Through extensive microbial-mammalian co-metabolism, the intestinal microbiota have evolved to exert a marked influence on health and disease via gut-brain-microbiota interactions. In this addendum, we summarize the findings of our recent study on the fecal microbiota and metabolomes of children with pervasive developmental disorder-not otherwise specified (PDD-NOS) or autism (AD) compared with healthy children (HC). Children with PDD-NOS or AD have altered fecal microbiota and metabolomes (including neurotransmitter molecules). We hypothesize that the degree of microbial alteration correlates with the severity of the disease since fecal microbiota and metabolomes alterations were higher in children with PDD-NOS and, especially, AD compared to HC. Our study indicates that the levels of free amino acids (FAA) and volatile organic compounds (VOC) differ in AD subjects compared to children with PDD-NOS, who are more similar to HC. Finally, we propose a new perspective on the implications for the interaction between intestinal microbiota and AD.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with multifactorial etiology, characterized by impairment in two main functional areas: (1) communication and social interactions, and (2) skills, interests and activities. ASD patients often suffer from gastrointestinal symptoms associated with dysbiotic states and a “leaky gut.” A key role in the pathogenesis of ASD has been attributed to the gut microbiota, as it influences central nervous system development and neuropsychological and gastrointestinal homeostasis through the microbiota–gut–brain axis. A state of dysbiosis with a reduction in the Bacteroidetes/Firmicutes ratio and Bacteroidetes level and other imbalances is common in ASD. In recent decades, many authors have tried to study and identify the microbial signature of ASD through in vivo and ex vivo studies. In this regard, the advent of metabolomics has also been of great help. Based on these data, several therapeutic strategies, primarily the use of probiotics, are investigated to improve the symptoms of ASD through the modulation of the microbiota. However, although the results are promising, the heterogeneity of the studies precludes concrete evidence. The aim of this review is to explore the role of intestinal barrier dysfunction, the gut–brain axis and microbiota alterations in ASD and the possible role of probiotic supplementation in these patients.

A supervised multivariate model to classify the metabolome alterations between autistic spectrum disorders (ASD) patients and controls, siblings of autistic patients, has been realized and used to realize a network model of the ASD patients' metabolome. In our experiment we propose a quantification of urinary metabolites with the Mass Spectroscopy technique couple to Gas Chromatography. A multivariate model has been used to extrapolate the variables of importance for a network model of interaction between metabolites. In this way we are able to propose a network-based approach to ASD description. Children with autistic disease composing our studied population showed elevated concentration of several organic acids and sugars. Interactions among diet, intestinal flora and genes may explain such findings. Among them, the 3-(3-hydroxyphenyl)-3-hydroxypropanoic acid has been previously described as altered in autistic subjects. Other metabolites increased are 3,4-dihydroxybutyric acid, glycolic acid and glycine, cis-aconitic acid; phenylalanine, tyrosine, p-hydroxyphenylacetic acid, and homovanillic acid are all involved in the tyrosine pathway leading to neurotransmitter cathecolamine. GC-MS-based metabolomic analysis of the urinary metabolome suggests to have the required sensitivity and specificity to gain insight into ASD phenotypes and aid a personalized network-based medicine approach.