Fecal microbiota and metabolome of children with autism and pervasive developmental disorder not otherwise specified

Abstract

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.

Figure 1. Total and active bacteria found in feces of children.

Relative abundance (%) of total bacterial composition (16S-rDNA) (A) and metabolic active bacteria (16S-rRNA) (B) at the phylum level found in the fecal samples of Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), autistic (AD) and healthy (HC) children.

Figure 2. Principal component analysis (PCA) of active bacteria genera found in feces of children.

Score plot of the three principal components (PC) after PCA of the total bacterial genera information (16S-rRNA) for Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), autistic (AD) and healthy (HC) children. 1-10, number of fecal samples for each group of children.

Figure 3. Permutation analysis of the active bacterial genera found in feces of children.

Metabolically active bacterial genera composition found in Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), autistic (AD) and healthy (HC) children. Double dendrogram representation of clustering was performed. The genera that showed values ​​less than 0.1% of the total metabolically active bacterial were grouped together on the same phylum (others Actinobacteria, others Bacteroidetes, others Firmicutes, others Proteobacteria). 1-10, number of fecal samples for each group of children.

Figure 4. Fecal levels of free amino acids (FAA) in children.

Concentration (mg/kg) of FAA found in fecal samples of Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), autistic (AD) and healthy (HC) children. Data are the means of three independent experiments and standard deviations, performed in duplicate (n=6).

Figure 5. Canonical Discriminant Analysis of Principal Coordinates (CAP) of volatile organic metabolites in feces of children.

Loading coefficient plots of the volatile organic compounds from fecal samples of Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) (A), autistic (AD) (B) and healthy (HC) children. Compounds significantly associated with the feces of PDD-NOS children (negative axis) or HC children (positive axis) Data are the means of three independent experiments (n = 3).

Figure 6. Principal component analysis (PCA) of volatile organic metabolites found in feces of children.

Score plots of the two principal components (PC) after PCA of volatile organic metabolites found in fecal samples of Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS), autistic (AD) and healthy (HC) children. All the variables used were listed in Table S1.