Mucosal and salivary microbiota associated with recurrent aphthous stomatitis

Abstract

Background Recurrent aphthous stomatitis (RAS) is common oral ulcerative disease to all ages. RAS is characterized by recurrent occurrence of ulceration which is extremely painful and heal slower than traumatic ulcers. Many factors, including stress, hormonal imbalance, oxidative stress, genetic predisposition, viral and bacterial infection, food allergies, vitamin and microelement deficiencies, have been suggested to contribute to the occurrence of RAS. However, the etiopathogenesis of RAS is unclear.

Methods The bacterial communities of the oral mucosa and saliva from RAS patients with active lesions (RAS, n = 18 for mucosa and n = 8 for saliva) and control subjects (n = 18 for mucosa and n = 7 for saliva) were analyzed by pyrosequencing of the 16S rRNA genes. The species richness and diversity index were calculated using the Ribosomal RNA database projects pyrosequencing pipeline (http://pyro.cme.msu.edu). The overall phylogenetic distance between communities was estimated using the weighted Fast UniFrac and was visualized using PCoA. Immortalized human oral keratinocyte HOK-16B cells originated from the retromolar gingival tissue were maintained in keratinocyte growth-culture medium (Clonetics, San Diego, CA, USA) containing supplementary growth factors. HOK-16B cells plated into 48-well plates at 4x104 cells/well in triplicate were cultured for 24 h and then infected with bacteria at the multiplicity of infection (MOI) 0, 100, 500, and 1000. The viability and total number of live cells in each well were determined by trypan blue exclusion under a microscope. Results There were no significant differences in the richness and diversity of the mucosal and salivary microbiota between the controls and the RAS. However, the mucosal microbiota of the RAS patients showed increased inter-subject variability. A comparison of the relative abundance of each taxon revealed decreases in the members of healthy core microbiota but increases of rare species in the mucosal and salivary microbiota of RAS patients. Particularly, decreased Streptococcus salivarius and increased Acinetobacter johnsonii in the mucosa were associated with RAS risk. A dysbiosis index, which was developed using the relative abundance of A. johnsonii and S. salivarius and the regression coefficients, correctly predicted 83 % of the total cases for the absence or presence of RAS. Interestingly, A. johnsonii substantially inhibited the proliferation of gingival epithelial cells and showed greater cytotoxicity against the gingival epithelial cells than S. salivarius.

Conclusion Pyrosequencing analysis successfully characterized the oral microbiota of RAS patients compared with healthy controls up to the species level. The mucosal microbiota of RAS lesions are characterized as decreases in the members of healthy core microbiota but increases of rare species, and a decrease in S. salivarius and an increase in A. johnsonii are associated with RAS risk. These findings may provide a diagnostic tool and new targets for the therapeutic. management of RAS.