High-Throughput Sequencing of Oral Microbiota in Candida Carriage Sjögren's Syndrome Patients: A Pilot Cross-Sectional Study

Abstract

Background: This study sought to characterize the saliva microbiota of Candida carriage Sjögren's syndrome (SS) patients compared to oral candidiasis and healthy patients by high-throughput sequencing.

Methods: Fifteen patients were included, with five Candida carriage SS patients (decayed, missing, and filled teeth (DMFT) score 22), five oral candidiasis patients (DMFT score 17), and five caries active healthy patients (DMFT score 14). Bacterial 16S rRNA was extracted from rinsed whole saliva. PCR amplification generated DNA amplicons of the V3-V4 hypervariable region, which were sequenced on an Illumina HiSeq 2500 sequencing platform and compared and aligned to the SILVA database. Taxonomy abundance and community structure diversity was analyzed using Mothur software v1.40.0.

Results: A total of 1016/1298/1085 operational taxonomic units (OTUs) were obtained from SS patients/oral candidiasis patient/healthy patients. Treponema, Lactobacillus, Streptococcus, Selenomonas, and Veillonella were the primary genera in the three groups. The most abundant significantly mutative taxonomy (OTU001) was Veillonella parvula. Microbial diversity (alpha diversity and beta diversity) was significantly increased in SS patients. ANOSIM analyses revealed significantly different microbial compositional heterogeneity in SS patients compared to oral candidiasis and healthy patients.

Conclusion: Microbial dysbiosis differs significantly in SS patients independent of oral Candida carriage and DMFT.

Keywords: Sjögren’s syndrome; dental caries; high-throughput nucleotide sequencing; saliva microbiota.

Figure 1

Species profiling histogram showing the 10 most predominant genera of saliva in Test group (A01–A05)/Group P (B01–B05)/Group N (C01–C05). The abundance of the total sequences of the five most predominant genera in Test group/Group P/Group N were Treponema (10.2%/9.9%/10.2%), Lactobacillus (9.5%/10%/8.4%), Streptococcus (7.6%/7.9%/8.5%), Selenomonas (8.6%/7.1%/7.8%), and Veillonella (7.9%/7.1%/7.8%). The numbers and proportions of sequences identified at the genus level were comparable among the three groups (p > 0.05).

Figure 2

Rarefaction curves showing that sequencing reached saturation.

Figure 3

Five OTUs with mean abundance of taxonomy >0.001, displayed significant changes among all three groups: OTU001, genera Veillonella; OTU011, genera Streptococcus; OTU021, genera Veillonella; OTU045, genera Gemella; and OTU047, genera Anaeroglobus. * significantly different (p < 0.05); ** extremely significantly different (p < 0.01).

Figure 4

Alpha diversity (Ace, Chao, Shannon, and Simpson indices) of the three groups at the genus level. Microbiota community richness was analyzed by the ACE and Chao indices. Community diversity was analyzed by the Shannon and Simpson indices. Box plots indicate medium, minimum, and maximum values. Significant differences among three groups were evident for the four indices (p < 0.05).

Figure 4

Alpha diversity (Ace, Chao, Shannon, and Simpson indices) of the three groups at the genus level. Microbiota community richness was analyzed by the ACE and Chao indices. Community diversity was analyzed by the Shannon and Simpson indices. Box plots indicate medium, minimum, and maximum values. Significant differences among three groups were evident for the four indices (p < 0.05).

Figure 5

Beta diversity evident in principal coordinate analysis (PCoA) based on weighted Unifrac matrix. Similarity in bacterial composition is indicated by the distance between dots, with smaller distance denoting increased similarity. The spatial distribution of plots from Group P and N subjects overlapped, suggesting a similar set of genera. Plots from the Test group of subjects formed a cluster, having roughly greater values than the plot clusters from Group P and N subjects. Test group samples displayed a trend of dispersion along the PCoA2 axis. Group P and N samples displayed a trend of dispersion along the PCoA1 axis.