Microbiological and bioinformatics analysis of primary Sjögren's syndrome patients with normal salivation^§

Abstract

BackgroundReduced salivation is considered a major clinical feature of most but not all cases of primary Sjögren’s syndrome (pSS). Reduced saliva flow may lead to changes in the salivary microbiota. These changes have mainly been studied with culture that typically recovers only 65% of the bacteria present.ObjectiveThis study was to use high throughput sequencing, covering both cultivated and not-yet-cultivated bacteria, to assess the bacterial microbiota of whole saliva in pSS patients with normal salivation.MethodsBacteria of whole unstimulated saliva from nine pSS patients with normal salivation flow and from nine healthy controls were examined by high throughput sequencing of the hypervariable region V1V2 of 16S rRNA using the 454 GS Junior system. Raw sequence reads were subjected to a species-level, reference-based taxonomy assignment pipeline specially designed for studying the human oral microbial community. Each of the sequence reads was BLASTN-searched against a database consisting of reference sequences representing 1,156 oral and 12,013 non-oral species. Unassigned reads were then screened for high-quality non-chimeras and subjected to de novo species-level operational taxonomy unit (OTU) calling for potential novel species. Downstream analyses, including alpha and beta diversities, were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline. To reveal significant differences between the microbiota of control saliva and Sjögren’s saliva, a statistical method introduced in Metastats www.metastats.cbcb.umd.edu was used.ResultsSaliva of pSS patients with normal salivation had a significantly higher frequency of Firmicutes compared with controls (p=0.004). Two other major phyla, Synergistetes and Spirochaetes, were significantly depleted in pSS (p=0.001 for both). In addition, we saw a nearly 17% decrease in the number of genera in pSS (25 vs. 30). While Prevotella was almost equally abundant in both groups (25% in pSS and 22% in controls), about a twofold increase in pSS of Streptococcus (28% vs. 17%) and Veillonella (26% vs. 12%) was detected. Prevotella melaninogenica was the major species in controls (13%) while Veillonella atypica and the Veillonella parvula groups dominated in patient samples (14 and 14%). The scarcity in bacterial species in pSS compared with controls was also demonstrated by alpha and beta diversity analyses, as well as read abundance depicted in a phylogenetic tree.ConclusionsWhile Firmicutes was significantly higher in pSS patients than in controls, Synergistetes and Spirochaetes were significantly lower. The number of bacterial genera and species was also lower. These data showed that microbial dysbiosis is another key characteristic of pSS whole saliva which can occur independent of hyposalivation.

Oral microbiota in autoimmune polyendocrine syndrome type 1

“…Sample processing was done as previously described [20,26]. In summary, DNA was extracted from a 250 µl sample volume using the MasterPure™ DNA Purification kit (Epicentre, Illumina Company, Madison, WI) and was dissolved in 45 µl 1 × TE buffer.…”

“…The 16S rRNA hypervariable region V1-V2 was amplified in three parallel PCRs and sequenced on a 454 GS Junior System (Roche, Branford, CT). Primer sequences and amplifications reactions are given previously [20]. DNA quality and concentrations were assessed with Bioanalyzer 2100 (Agilent, Santa Clara, CA) and Nanodrop 3300 Flurospectrometer (Thermo Scientific, Wilmington, DE), all within the range to perform high throughput sequencing.…”

“…Bioinformatics analysis of sequence reads was executed as described previously [20]. In brief, raw sequence reads were subjected to a species-level, reference-based taxonomy assignment especially designed for studying the human oral microbial community [20].…”

“…In brief, raw sequence reads were subjected to a species-level, reference-based taxonomy assignment especially designed for studying the human oral microbial community [20]. The set of 16S rRNA reference sequences previously published by Al-Hebshi et al [27] and the NCBI 16S rRNA reference sequence set (ftp://ftp.ncbi.nlm.nih.gov/blast/db/16SMicrobial.tar.gz) were combined giving reference sequences representing a total of 1,151 oral and 12,013 non-oral species that were BLASTN-searched for each of the sequence reads.…”

“…Reduced salivary flow rate changes the oral microbiota [18] and in a study of patients with severe Sjögren’s syndrome Streptococcus salivarius, Neisseria pharyngis, Veillonella species, and Micrococcus mucilaginosus were reduced and the number of Staphylococcus aureus and Candida species were increased compared to healthy controls [19]. Another recent study of primary Sjögren’s syndrome patients with normal salivary flow rate found that the number of bacterial genera and species was lower in patients, and concluded that saliva dysbiosis is a key characteristic of primary Sjögren’s syndrome [20]. Moreover, changes in the oral microbiota are found to be associated with several other diseases including squamous cell carcinoma, atherosclerosis, bacteraemia, and rheumatoid arthritis [21].…”