Salivary microbiota and metabolome associated with celiac disease

Abstract

This study aimed to investigate the salivary microbiota and metabolome of 13 children with celiac disease (CD) under a gluten-free diet (treated celiac disease [T-CD]). The same number of healthy children (HC) was used as controls. The salivary microbiota was analyzed by an integrated approach using culture-dependent and -independent methods. Metabolome analysis was carried out by gas chromatography-mass spectrometry-solid-phase microextraction. Compared to HC, the number of some cultivable bacterial groups (e.g., total anaerobes) significantly (P < 0.05) differed in the saliva samples of the T-CD children. As shown by community-level catabolic profiles, the highest Shannon's diversity and substrate richness were found in HC. Pyrosequencing data showed the highest richness estimator and diversity index values for HC. Levels of Lachnospiraceae, Gemellaceae, and Streptococcus sanguinis were highest for the T-CD children. Streptococcus thermophilus levels were markedly decreased in T-CD children. The saliva of T-CD children showed the largest amount of Bacteroidetes (e.g., Porphyromonas sp., Porphyromonas endodontalis, and Prevotella nanceiensis), together with the smallest amount of Actinobacteria. T-CD children were also characterized by decreased levels of some Actinomyces species, Atopobium species, and Corynebacterium durum. Rothia mucilaginosa was the only Actinobacteria species found at the highest level in T-CD children. As shown by multivariate statistical analyses, the levels of organic volatile compounds markedly differentiated T-CD children. Some compounds (e.g., ethyl-acetate, nonanal, and 2-hexanone) were found to be associated with T-CD children. Correlations (false discovery rate [FDR], <0.05) were found between the relative abundances of bacteria and some volatile organic compounds (VOCs). The findings of this study indicated that CD is associated with oral dysbiosis that could affect the oral metabolome.

FIG 1

CLCP indices (utilization pattern substrate [H′], substrate richness [S], and equitability [E]) of the salivary microbiota of children with celiac disease who were treated with a gluten-free diet for at least 2 years (T-CD children) and healthy children (HC). Data are the means of three independent experiments (n = 3). The center line of each box represents the median, and the top and bottom of the box represent the 75th and 25th percentiles of the data, respectively. The top and bottom of the error bars represent the 5th and 95th percentiles of the data, respectively. The circles in each box plot extend to the outliers of the data.

FIG 2

Relative abundance (percent) of total bacteria, which were found at the phylum level in the saliva of T-CD children and healthy children.

FIG 3

Average number of species (OTUs), richness (Chao1), and diversity (Shannon index) values for the saliva of T-CD children and HC. The center line of each box represents the median, and the top and bottom of the box represent the 75th and 25th percentiles of the data, respectively. The top and bottom of the error bars represent the 5th and 95th percentiles of the data, respectively. The circles and asterisks in each box plot extend to the outliers and extremes of the data, respectively.

FIG 4

Principal-coordinate analysis based on weighted UniFrac analysis of all 16S rRNA gene sequences found in the saliva of T-CD children and HC.

FIG 5

Relative proportions (percent) of predominant bacteria, showing significant (P < 0.05) differences between the saliva samples of T-CD children and those of HC.

FIG 6

CAP loading coefficient plot (A) and score plot (B) of the volatile organic compounds found in saliva of T-CD children and HC. n.d., not defined.