Comparison of fecal secretory IgA concentrations and bacterial diversity between BD patients and normal individuals.

<p>(A) We evaluated the secretory IgA (sIgA) concentrations of fecal supernatants using ELISA. We observed a significant increase in sIgA concentrations of patients with BD (BD) compared with those of normal individuals (NI). (B) We counted OTU numbers (annotated species numbers) and estimated alpha diversity score (Chao 1 and Shannon indexes) of each sample. We compared the titers between BD patients and normal individuals. We did not find significant differences in the parameters between BD patients and normal individuals. These biological parameters of BD patients and normal individuals were displayed with dot plots. A box-plot and a mean level (green line) of each group of BD patients and normal individuals were indicated. (C) We estimated beta diversity between BD patients and normal individuals using PCoA of QIIME software with linear conversion formulas. We visualized the PCoA plots in a three dimensional structure where three axes and each contribution ratio (principal coordinate, PC1–3, %) were depicted. We calculated the distance between the distribution of BD patients and that of normal individuals using a two-sided Student's two-sample t-test as an exploratory analysis. We obtained a significant P value of the test of beta diversity between BD patients and normal individuals.</p

Comparison of the taxa showing different abundance values in BD patients and normal individuals.

<p>We analyzed the metagenomic data of bacterial taxa using LEfSe to detect major differences between BD patients (BD) and normal individuals (NI). The LEfSe provides us with cladograms of six-level (from kingdom to genus). Significantly enriched bacterial taxa in samples obtained from BD patients were demonstrated using red circles and shadings. Significantly enriched bacterial taxa in samples obtained from normal individuals were demonstrated using green circles and shadings. In patients with BD, the phylum <i>Actinobacteria</i>, namely the classes <i>Actinobacteria</i> and <i>Coriobacteria</i>, the orders <i>Bifidobacteriales</i> and <i>Coriobacteriales</i>, and the genera <i>Bifidobacterium</i>, <i>Eggerthella</i> and <i>Atopobium</i> had large effect sizes. The phylum <i>Firmicutes</i>, the class <i>Clostridia</i>, the order <i>Clostridiales</i>, the family <i>Veillonellaceae</i>, and the genera <i>Megamonas</i> and <i>Phascolarctobacterium</i> had large effect sizes in normal individuals.</p

Direct comparison of bacterial taxonomic abundance between BD patients and normal individuals.

<p>We compared directly the relative abundance (expressed as parts per unit) between BD patients (BD) and normal individuals (NI). We conducted Wilcoxon rank sum test for the differences in every taxon, followed by Tukey’s honestly significant difference (HSD) test. We considered that bacterial taxa increased or decreased significantly in patients with BD as compared with those in normal individuals by fulfilling the following criteria simultaneously; #1: bacterial taxa showing significant differences by the Wilcoxon rank sum test, and #2: bacterial taxa showing positive Tukey’s HSD values. We found that there were significant differences in 11 bacterial taxa (also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153746#pone.0153746.g002" target="_blank">Fig 2</a> with asterisks). “p__”, “c__”, “o__”, “f__” and “g__” indicate phylum, class, order, family and genus, respectively. Relative abundance of bacterial taxa in BD patients and normal individuals were displayed with dot plots. A box-plot and a mean level (green line) of each group of BD patients and normal individuals were indicated.</p

Demographical and clinical characteristics of patients with Behcet’s disease and normal individuals.

<p>Demographical and clinical characteristics of patients with Behcet’s disease and normal individuals.</p