The community of microorganisms that exist symbiotically within our bodies is called the microbiota. The number of bacterial cells in our body is the same or may exceed the number of human cells [1]. During 2005–2010, classical Sanger sequencing was used to examine the diversity of the oral microbiota. Studies have reported that the majority of microbes present in the oral cavity belonged to the following six phyla: Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, Fusobacteria, and Spirochetes [32, 33]. In this study, using next-generation sequencing and metagenomic analysis, we identified 13 phyla, 92 genera, and 410 bacterial species in five oral plaque specimens. In line with previous study findings, Bacteroidetes (29.1%), Firmicutes (25.4%), Actinobacteria (16.6%), Proteobacteria (13.2%), Fusobacteria (4.3%), and Spirochetes (1.2%), which accounted for 89.9% of the total bacterial phyla, were detected in our study.
The oral cavity contains a very large variety of bacterial species as it is exposed to the external environment and because of the presence of various niche habitats, including hard and soft tissues, in the complex intraoral environment. Segata et al. compared the bacterial composition of specimens from different parts of the oral cavity of healthy individuals and identified the following parts where oral bacterial communities are formed: 1) tooth surface (supragingival plaque and subgingival plaque), 2) dorsum of the tongue (classified into the pharynx, palatine tonsils, and saliva), 3) buccal mucosa, keratinized gingiva, and hard palate [34]. According to their report, Actinobacteria, Gammaproteobacteria, Betaproteobacteria, and Flavobacteriia are hallmark classes of supragingival plaque. Consistent with their findings, we detected Actinobacteria (average detection rate, 1.43E-01%), Gammaproteobacteria (2.40E-02%), Betaproteobacteria (8.32E-02%), and Flavobacteriia (5.00E-02%).
Dental and other procedures that commonly cause bleeding, such as tooth extraction, periodontal surgery, scaling, and root planning, are assumed to contribute to the development of bacteremia [12]. Bacteremia has also been reported to occur after routine oral hygiene procedures such as brushing (0%–26%), flossing (20%–58%), and even chewing (17%–51%) [13, 14]. We identified 10 bacterial phyla, 69 bacterial genera, and 219 bacterial species in 15 aortic valve specimens. In particular, nine bacterial species (3.1%) were found to be in common between the oral plaque and aortic valve tissue samples (Fig. 2). Among these nine species, S. oralis, S. sanguinis, and S. pneumoniae belong to the mitis group of the genus Streptococcus. In previous studies involving 239 cases of infective endocarditis, mitis group species were identified in 61 (25.5%) of 100 (41.8%) cases in which oral streptococcus was the causative organism [35–37]. Our results also demonstrate that various bacterial species, including oral streptococci, can frequently enter the bloodstream and colonize and invade cardiovascular tissues, exerting various effects.
Aortic stenosis is a pathological condition marked by the infiltration of inflammatory macrophages and the formation of extracellular matrix in the valve tissue. As these pathological conditions progress, the valve cusps thicken and harden, and mineral deposition occurs [38]. Anatomically, the aortic valve comprises three layers: the collagen-rich fibrosa layer on the aortic side of the valve, the glycosaminoglycan-rich spongiosa layer in the middle, and the elastin-rich ventricularis layer on the left ventricular side [39]. The onset site of aortic valve calcification in this three-layer structure is thought to be proximal to the base of the fibrosa layer, which is in contact with the aortic wall, and calcification progresses from the base to the tip [40]. Additionally, pathological conditions or external stimulations promote the production of cytoskeletal or osteogenic proteins, which in turn induce the differentiation of valvular interstitial cells, which make up the aortic valve, into myofibroblasts and osteoblasts. It has been suggested that myofibroblasts promote fibrosis and sclerosis of the valve tissue, whereas osteoblasts promote the deposition of mineral components such as calcium [41]. However, the activation mechanism for cells with these varying properties in vivo has not been elucidated, and it is unknown whether the activation of these cells contributes to valve dysfunction. Oral streptococcal virulence factors include surface protein antigen I/II, serotype-specific polysaccharides, and lipoteichoic acid, all of which have been shown to induce inflammatory responses in human cells [42]. Streptococcus oralis expresses coaggregation receptor polysaccharide (RPS) that functions as a receptor for surface adhesins during symbiosis with other bacterial species, facilitating the formation of an oral biofilm. Recently, RPS has been reported to induce an inflammatory response in human aortic endothelial cells, leading to an increased production of cytokines, adhesion molecules, and TLR-2 [43]. Thus, these molecular mechanisms may be involved in the phenotype associated with valvular interstitial cells and the pathogenesis of aortic stenosis.
Based on the characteristics of the microbiota, the aortic valve tissue samples were divided into two groups, groups A and B (Fig. 4). Nocardia cyriacigeorgica was the most abundant bacterial species in group B (Table 1). The genus Nocardia comprises aerobic, gram-positive, catalase-positive, and partial mycobacterial filamentous bacteria that exist in the soil and decaying plants and belong to the order Actinomycetes. Their transmission most commonly occurs via inhalation, and other potential routes include penetrating injuries, iatrogenic procedures, and food ingestion [44]. The risks for infection with Nocardia spp. include comorbidities such as diabetes, chronic lung disease, alcoholism, and active cancer, as well as immunocompromised conditions such as immunosuppressive therapy, and endovascular device placement [45–47]. Although an association with pre-existing diseases, such as diabetes, could not be verified in group B, the bacteria in this group were significantly correlated with low levels of serum albumin (p = 0.0026). The occurrence of different forms of immunodeficiency and malnutrition in individuals in group B may have been associated with the presence of N. cyriacigeorgica.
Recent epidemiological, clinical, and experimental analyses have supported the relationship between bacteremia or inflammation due to periodontal disease and systemic disease [48]. To the best of our knowledge, this study is the first to comprehensively characterize the composition of oral plaque and aortic valve microbiota in patients with aortic stenosis. Using metagenomic analysis, we identified bacterial species shared between the oral cavity and aortic valve tissues. Our results support that bacterial species that form the oral microbiota may translocate to the aortic valve due to bacteremia in patients with aortic stenosis and thus may be associated with disease onset and severity. However, in this study, it was not possible to verify the mechanism by which bacteria attach to the aortic valve to affect the pathology of aortic stenosis. Additionally, owing to the high cost of metagenomic analysis ($830 per case) and occasional urgent need for surgery for aortic stenosis, only a limited number of samples from patients who provided consent was available. Despite this limitation, crucial events associated with the development of bacteremia in the pathology of aortic stenosis were adequately captured. It is sufficiently clear that the management of periodontal disease and proper oral care can positively affect the morbidity, mortality, and health care costs associated with nonoral systemic diseases. Further studies aimed at developing strategies for maintaining the integrity of the oral microbiota are warranted.