PLS-DA models based on the similarity distance matrix of DGGE built as a function of the nucleic acids.

<p>Data from DNA (blue) and RNA (yellow).</p

Fecal Microbiota and Metabolome of Children with Autism and Pervasive Developmental Disorder Not Otherwise Specified

<div><p>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 (<i>P</i><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, <i>Faecalibacterium</i> and <i>Ruminococcus</i> were present at the highest level in fecal samples of PDD-NOS and HC children. <i>Caloramator</i>, <i>Sarcina</i> and <i>Clostridium</i> 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 <i>Eubacterium siraeum</i>, the lowest level of Eubacteriaceae was found on fecal samples of AD children. The level of Bacteroidetes genera and some <i>Alistipes</i> and <i>Akkermansia</i> 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, <i>Bifidobacterium</i> 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.</p> </div

Microbial species identification after band sequencing of the variable V3 region of the 16S rRNA gene.

<p>^<b>§</b>The bands are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128669#pone.0128669.g006" target="_blank">Fig 6</a>, Plot C.</p><p>Dietary habit-specific bands were selected and subjected to identification.</p

PLS-DA models based on the RNA-DGGE similarity distance matrix from each diet group using the geographical site as the discriminating factor.

<p>Plot A, omnivore individuals; Plot B, ovo-lacto-vegetarian individuals; Plot C, vegan individuals. The samples are color coded according to the geographical site (Bari: silver; Bologna: gold; Parma: blue; Torino: maroon).</p

RNA-DGGE band distribution.

<p>Plot A and Plot B, Venn diagrams of the unique and overlapping bands present in the RNA-DGGE profile of the omnivore (red), ovo-lacto-vegetarian (green) and vegan (blue) individuals for the V3 and V9 regions, respectively; Plot C, RNA-DGGE fingerprints of the V3 amplicons, where the letters indicate the dietary groups: the omnivore (O), ovo-lacto-vegetarian (VG) and vegan (V) band identifications are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128669#pone.0128669.t002" target="_blank">Table 2</a>; Plot D, RNA-DGGE fingerprints of the V9 amplicons; the band identifications are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128669#pone.0128669.t003" target="_blank">Table 3</a>.</p

PLS-DA models based on the RNA-DGGE similarity distance matrix as a function of the geographical site.

<p>The plot is color coded as a function of the city: Bari (silver), Bologna (gold), Parma (blue), Torino (maroon).</p

Distribution of the viable counts of the three diets according to the different culture media.

<p>Box plot interpretation: the central line indicates the median, upper and lower box lines of the first and third quartiles (Q1 and Q3), respectively, and the bars at the end of the whiskers represent the distribution extremities, while the dots indicate outliers. Plot A, all the media considered together; Plot B, GSP medium at 25°C; Plot C, Chromocult Coliform Agar at 37°C; Plot D, Bifidumbacterium Agar at 37°C; Plot E, MRS at 25°C; Plot F, MRS at 37°C; Plot G, Wilkins-Chalgren Anaerobe Agar + GN Supplement; Plot H, Bacteroides Bile Esculine Agar. For specifications on the counted microbial groups, reference can be made to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128669#pone.0128669.t001" target="_blank">Table 1</a>.</p

Principal component analysis (PCA) of volatile organic metabolites found in feces of subjects.

<p>Score plots of the two principal components (PC) after principal component analysis (PCA) of volatile organic metabolites of the fecal (A) and urine (B) samples of immunoglobulin A nephropathy (IgAN) non progressor (NP) and progressor (P) patients, and healthy controls (HC).</p

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

<p>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).</p

Total and active bacteria found in feces of children.

<p>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. </p