Kim et al. BMC Microbiology (2016) 16:57 DOI 10.1186/s12866-016-0673-z RESEARCH ARTICLE Open Access Mucosal and salivary microbiota associated with recurrent aphthous stomatitis Yun-ji Kim1, Yun Sik Choi1, Keum Jin Baek1, Seok-Hwan Yoon2, Hee Kyung Park3* and Youngnim Choi1* Abstract Background: Recurrent aphthous stomatitis (RAS) is a common oral mucosal disorder of unclear etiopathogenesis. Although recent studies of the oral microbiota by high-throughput sequencing of 16S rRNA genes have suggested that imbalances in the oral microbiota may contribute to the etiopathogenesis of RAS, no specific bacterial species associated with RAS have been identified. The present study aimed to characterize the microbiota in the oral mucosa and saliva of RAS patients in comparison with control subjects at the species level. Results: 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. There were no significant differences in the alpha diversity between the controls and the RAS, but 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: RAS is associated with dysbiosis of the mucosal and salivary microbiota, and two species associated with RAS have been identified. This knowledge may provide a diagnostic tool and new targets for therapeutics for RAS. Keywords: Recurrent aphthous stomatitis, Oral microbiota, Pyrosequencing Background suggested to trigger or to be associated with RAS [3]. How- Recurrent aphthous stomatitis (RAS) is one of the most ever, the etiopathogenesis of RAS remains unclear. Conse- common oral mucosal disorders affecting at least 10 to quently, no curative treatment is available and patient care 20 % of the general population [1]. RAS is characterized by primarily consists of symptomatic treatment [1]. the recurrent occurrence of well-circumscribed, single or Among the bacterial infections, the role of a Strepto- multiple ulcers that are extremely painful and heal more coccus strain (first identified as S. sanguinis but now re- slowly than traumatic ulcers of similar size [2]. Diverse fac- classified as S. oralis) has been extensively studied since tors, including genetic predisposition, immunologic distur- its isolation from a RAS lesion. Cross-reaction of anti- bances, viral and bacterial infections, food allergies, vitamin Streptococcal antibodies with autoantigens in the oral and microelement deficiencies, systemic diseases, hormonal mucosa was proposed as an etiopathogenic mechanism imbalance, mechanical injuries, and stress, have been of RAS, but no evidence to support this hypothesis has been found [4]. Later, lack of association between S. ora- * Correspondence: [email protected]; [email protected] 3Department of Oral Medicine and Oral Diagnosis, School of Dentistry and lis and RAS was reported based on the less frequent Dental Research Institute, Seoul National University, 101 Daehak-ro, Seoul, detection of S. oralis in RAS than in healthy control Jongno-gu 110-744, Republic of Korea samples [5]. Cross-reactive recognition of the mycobac- 1Department of Oral Microbiology and Immunology, School of Dentistry and Dental Research Institute, Seoul National University, 101 Daehak-ro, Seoul, terial and human heat shock protein 65-60 antigen by T Jongno-gu 110-744, Republic of Korea cells of RAS patients has been reported, suggesting the Full list of author information is available at the end of the article © 2016 Kim et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. BMC Microbiology (2016) 16:57 Page 2 of 10 role of Mycobacterium tuberculosis and autoreactive T sampling sites of RAS lesions included the tip of the cells in RAS [6]. Accumulated evidence supports the as- tongue (n = 4), the buccal mucosa (n = 4), and the labial sociation between RAS and Helicobacter pylori infection, mucosa (n = 10). Twelve patients had a single ulcer and but the presence of H. pylori at the RAS lesions is con- six patients had multiple ulcers on the tip of the tongue, troversial [7, 8]. In an attempt to discover the micro- labial mucosa, and soft palate. The control group included organisms present in RAS lesions, Marchini et al. [9] eight males and 10 females with an age distribution of 21 studied the mucosal microbiota in RAS patients using a to 71 years. The mucosal sampling sites included the buc- culture-independent method. Due to limitations in the cal mucosa (n = 11) and the labial mucosa (n =7). methods available at that time, only 57 and 38 phylo- types were defined from 10 RAS and 10 healthy subjects, The alpha and beta diversities of the oral microbiota respectively. Recently, the salivary microbiota in patients From the 51 communities, total 484,501 filtered reads with inflammatory bowel disease, where RAS is one of (average 9500 reads per sample) with an average length the extraintestinal manifestations of inflammatory bowel of 479 bp were obtained, which resulted in greater than disease [10], and the oral mucosal microbiota in RAS pa- 99 % Good’s coverage for each sample. We first com- tients have been studied by high-throughput sequencing pared the alpha diversity between the control and RAS of the 16S rRNA genes [11, 12]. In addition, Terminal- group. The species richness of the RAS microbiota esti- Restriction Fragment Length Polymorphism analysis of mated by Chao 1 was not significantly different from bacterial 16S rRNA genes, the human oral microbe iden- that of controls either in the mucosa (314 ± 19 vs. 292 ± tification microarrays, and matrix-assisted laser desorp- 22) or in the saliva (377 ± 14 vs. 447 ± 33). The diversities tion/ionization time-of-flight analysis have been applied of RAS microbiota determined by the Shannon index were to study the oral microbiota of RAS patients [13, 14]. Al- also comparable to those of controls both in the mucosa though all previous studies suggested that imbalances in (3.57 ± 0.11 vs. 3.56 ± 0.06) and in the saliva (4.02 ± 0.09 the oral microbiota may be involved in the etiopathogen- vs. 4.21 ± 0.1) (Fig. 1a). esis of RAS, no specific bacterial species associated with UniFrac-based principal coordinate analysis (PCoA) to RAS have been identified. We previously characterized determine variation among the samples revealed that the the murine oral microbiota to the species level by pyrose- microbiota profile was differentiated better by the ana- quencing [15]. Therefore, this study aimed to characterize tomical sites, i.e., mucosal surfaces vs. saliva, than by dis- the microbiota of the oral mucosa and saliva of RAS pa- ease. The different locations in the mucosa, i.e., tongue tients compared with control subjects at the species level. tip, labial, or buccal, did not show distinct clustering Pyrosequencing analysis successfully characterized the (Fig. 1b). Although PCoA clustering did not reveal clear oral microbiota of RAS patients and identified two species separation between the control and RAS communities, associated with RAS risk. the intergroup UniFrac distance (0.061 ± 0.001) was higher than the intragroup distance of controls (0.057 ± 0.001), Results suggesting a significant difference in the bacterial profile Subjects between control and RAS samples. In addition, a higher The demographic data of RAS patients and control sub- intragroup UniFrac distance in the RAS (0.067 ± 0.001) jects included in the current study are summarized in compared to the control group suggested the increased Table 1. Nine males and nine females with the active le- inter-subject variability for RAS lesions (Fig. 1c). In the sions of minor RAS were included and the age distribu- salivary communities that reflect not only the diseased tion of the patients was 19 to 81 years. The mucosal sites but also the healthy sites of patients, no significant differences were observed in intra- or intergroup UniFrac Table 1 The demographic data of the control subjects and RAS distances. patients Control subjects (n = 18) RAS patients (n = 18) Differences in oral microbiota composition between the Gender 8 males, 10 females 9 males, 9 females control and RAS group Age 43.6 ± 3.7 43.8 ± 3.9 Next, the relative abundance of each taxon between the control and RAS group was compared. Although a total Ulcer - Single: 12 (66.7 %) numbers Multiple: 6 (33.3 %) of 26 different phyla were identified from the mucosa samples, Firmicutes, Proteobacteria, Actinobacteria, Bac- Sampling sites Lip labial mucosa: 7 (38.9 %), Lip labial mucosa: 10 (55.6 %), Buccal mucosa: 11 (61.1 %) Buccal mucosa: 4 (22.2 %) teroidetes, and Fusobacteria encompassed the majority Tonguetip:4(22.2%), of the sequences (>99 % in Controls and >97 % in RAS).