Next Article in Journal
Application of Real-Time PCR Syndromic Panel on Lower Respiratory Tract Samples: Potential Use for Antimicrobial De-Escalation
Previous Article in Journal
Pathogenicity in Chicken Anemia Virus with Eimeria tenella: Concurrent Co-Infection and Secondary Eimeria tenella Infection
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 13
Issue 7
10.3390/microorganisms13071677
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Oral Dysbiosis Is Associated with the Pathogenesis of Aortic Valve Diseases

by
Erika Yaguchi
1,†,
Yuske Komiyama
1,†,
Shu Inami
2,
Ikuko Shibasaki
3,
Tomoaki Shintani
4,
Ryo Shiraishi
1,
Toshiki Hyodo
1,
Hideki Shiba
5,
Shinsuke Hamaguchi
6,
Hirotsugu Fukuda
3,
Shigeru Toyoda
2,
Chonji Fukumoto
1,
Sayaka Izumi
1,
Takahiro Wakui
1,* and
Hitoshi Kawamata
1,*
1
Department of Oral and Maxillofacial Surgery, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Shimotsuga, Mibu 321-0293, Tochigi, Japan
2
Department of Cardiovascular Medicine, Dokkyo Medical University School of Medicine, 880 Kitakoba Yashi, Shimotsuga, Mibu 321-0293, Tochigi, Japan
3
Department of Cardiac and Vascular Surgery, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Shimotsuga, Mibu 321-0293, Tochigi, Japan
4
Center for Oral Clinical Examination, Hiroshima University Hospital; 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Hiroshima, Japan
5
Department of Biological Endodontics, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Hiroshima, Japan
6
Department of Anesthesiology, Dokkyo Medical University School of Medicine, 880 Kitakobayashi, Shimotsuga, Mibu 321-0293, Tochigi, Japan
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microorganisms 2025, 13(7), 1677; https://doi.org/10.3390/microorganisms13071677
Submission received: 5 June 2025 / Revised: 13 July 2025 / Accepted: 14 July 2025 / Published: 16 July 2025
(This article belongs to the Section Medical Microbiology)
Browse Figures
Review Reports Versions Notes

Abstract

The involvement of oral bacteria in the pathogenesis of distant organs, such as the heart, lungs, brain, liver, and intestine, has been shown. We analyzed the distribution of bacterial species in the resected aortic valve by 16S rRNA metagenomic analysis and directly compared their gene sequences with those in the oral cavity. Thirty-two patients with aortic stenosis or aortic regurgitation who underwent aortic valve replacement were enrolled in this study. Antibody titer against periodontal pathogenic bacteria in the patient’s serum was analyzed. The genetic background and distribution of bacterial species on subgingival plaque, the dorsal surface of the tongue, and the resected aortic valve were analyzed. Patients with aortic valve disease were shown to have more severe periodontal disease by the detection of antibodies against Socransky’s red-complex bacteria of periodontitis. Bacterial DNA was detected in the aortic valves of 12 out of 32 patients. The genomic sequences of the V3-V4 region of the 16S rRNA in some bacteria isolated from the aortic valves of six patients who underwent metagenomic analysis were identical to those found in the oral cavity. The findings indicate that bacteria detected in the aortic valve may be introduced through oral dysbiosis, a condition characterized by an imbalance in the oral microbiota that increases the risk of periodontal disease and dental caries. Oral dysbiosis and the resulting potential bacteremia are associated with the pathogenesis of aortic valve diseases.

1. Introduction

The involvement of oral bacteria in the pathogenesis of distant organs, such as the heart, lungs, brain, liver, and intestine, has been shown [1,2]. Dysbiosis is known as pathological changes (type and/or proportion of bacteria) of commensal bacteria in some organs, which cause various diseases [3]. Oral dysbiosis is a condition characterized by an imbalance in the oral microbiota that increases the risk of periodontal disease and dental caries. Recently, several reports have demonstrated an association between oral bacteria or oral dysbiosis and diseases of other organs through metagenome analysis and molecular biological analysis [4,5].
One known disease closely associated with oral bacteria is infectious endocarditis (IE), which causes verrucae on the endocardium. Aortic valve disease (AV) is known to increase the risk of IE. Among aortic valve diseases (aortic stenosis (AS) and aortic regurgitation (AR)), AS is a condition in which blood flow from the left ventricle to the ascending aorta during systole is obstructed due to narrowing of the aortic valve orifice [6]. The causes include idiopathic degenerative sclerosis with calcification (congenital bicuspid valves prone to sclerosis) and rheumatic fever. Valve replacement or valvuloplasty is indicated when stenosis and regurgitation after AS/AR are severe [7]. As per the general concept of the pathogenesis of IE, the blood flow jets caused by the valvular diseases (AS and AR) damage the valves and endocardium, resulting in the formation of a sterile vegetation at the site of the damage. Bacterial infection on these vegetations is thought to lead to the development of IE.
We have previously reported that more than 60% of bacteria detected in the blood cultures of patients with IE are recognized as oral bacteria [8]. In our previous study, bacterial cultures of blood from patients with IE were compared with dental plaque cultures in the oral cavity to verify the morphological and biochemical identity. DNA sequencing of bacteria in blood and dental plaque from patients with IE can reveal their genetic identity. However, in only one case the bacteria detected in dental plaque and blood had a 100% identical 16S rRNA gene sequence [8]. Although this study showed indirect evidence for so-called focal dental infections in distant organs, it was difficult to genetically identify the causative bacteria by comparing the limited number of clones detected in the bacterial cultures.
In this study, we detected bacteria in the resected aortic valve and analyzed the distribution of bacterial species in the resected aortic valve by metagenomic analysis. Simultaneously, we analyzed the distribution of bacterial species in subgingival dental plaque, which causes periodontal disease, and in swabs from the dorsal surface of the tongue, which reflects the bacterial microbiota of the oral cavity in the same patients. Then we verified whether oral bacteria reached the aortic valve by directly comparing their gene sequences.

2. Materials and Methods

2.1. Patients

Thirty-two patients with AS or AR who underwent aortic valve replacement between 1 May 2020, and 31 March 2021, at Dokkyo Medical University Hospital were enrolled in this study. The inclusion criteria for this study were that the aortic valve was resected during the designated period and the material was aseptically available. No exclusion criteria were defined. The 32 patients included 14 patients with diabetes mellitus who were adequately controlled. Four patients out of thirty-two were taking steroids to control their rheumatoid arthritis or interstitial pneumonia. None of the patients had received antimicrobial therapy in a week prior to oral bacteria collection or in a week prior to aortic valve resection. Severity of the periodontitis was classified according to the classification of periodontitis by the Japanese Society of Periodontology [9]. This study was approved by the Ethics Committee of Dokkyo Medical University (approval no. R-16-14J and R-37-20J). Patients were included in this study only when consent was obtained from the patient or the key person. In cases where the patients themselves could not make a decision, we explained the details of this research to the key person in this study. In this study, we obtained genetic information from microorganisms, but not human-derived information.

2.2. Measurement of Serum Antibody Titer Against Periodontal Pathogenic Bacteria

Serum antibody titers were measured according to a previous report [10,11]. Serum from the preoperative blood collection of patients was isolated and stored at −80 °C. Serum was collected from patients during surgery and stored at −80 °C. Serum IgG antibody titers against periodontal pathogenic bacteria were determined by the enzyme-linked immunosorbent assay (ELISA) using strains of bacteria that had already been isolated and identified. Sonicated preparations of bacteria (Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), Aggrigatibactor actinomycetemcomitans (Aa), Eikenella corrodens (Ec), Fusobacterium nucleatum (Fn), Prevotella intermedia (Pi), Prevotella nigrescens (Pn), Campylobactor rectus (Cr)) were used as bacterial antigens. The sera from five healthy subjects (ages 12–81) were pooled and used for calibration. With serial dilutions of this pooled control serum, the standard reaction was defined as an ELISA unit (EU), so that 100 EU corresponded to 1: 3200 dilutions of the calibrator sample. The following formula was applied to the EU to calculate the diagnostic standardized value: (IgG titer of patient- mean IgG titer of healthy subjects)/2 standard deviations (SD) determined by mean IgG titer of five healthy subjects [10].

2.3. DNA Extraction from the Aortic Valve or Oral Bacteria

Aortic valves were stored in 1.5 mL microtubes at −80 °C immediately after surgical excision. Isospin Tissue DNA (Nippon Gene, Co. Ltd., Tokyo, Japan) was used to extract DNA from excised aortic valves. Samples of the dorsal surface of the tongue, representing the bacterial microbiota of the oral cavity, were collected using cotton swabs. The swabs were rubbed on the dorsal surface of the tongue and stored in conical tubes at −80 °C until DNA extraction. In addition, subgingival plaque samples were collected from sites with obvious inflammatory conditions, such as periodontitis, using a manual scaler and stored in 1.5 mL microtubes at −80 °C until DNA extraction. DNA was extracted from plaque and tongue swab samples using the Isoil DNA extraction kit following the manufacturer’s instructions (Nippon Gene, Co. Ltd., Tokyo, Japan). The plaques were directly added to the extraction buffer, and the swab tips were cut into the extraction buffer, thoroughly agitated, and centrifuged to obtain the supernatant.

2.4. Confirmation of Bacterial DNA in the Extracted Samples

Extracted DNA was confirmed using primers against bacterial 16S rRNA. The primers used were 16S rRNA-27f: 5′-AGAGAGTTTGATCCTGGCTCAG-3′ and 16S rRNA-1492r: 5′-ACGGCTACCTTGTTACGACTT-3′. We performed PCR on extracted DNA samples using QUICK Taq HS DyeMix (TOYOBO Co., Ltd., Osaka, Japan). The PCR products were subjected to 1.2% agarose gel electrophoresis to detect a band of approximately 1500 bp corresponding to 16S rRNA.

2.5. 16S rRNA Gene Amplicon Sequencing Analysis

For amplicon sequencing, libraries were prepared according to the Illumina 16S metagenomic sequencing library preparation protocols. A total of 2.5 µL of microbial genomic DNA (5 ng/µL in 10 mM Microbial genomic DNA 2.5 µL (5 ng/µL in 10 mM Tris pH 8.5) was used for the first PCR with the following primers: forward, 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGAGACAGCCTAHGGGRBGCAGCAG-3′, and reverse, 5′-GTCGTGGGCTCTCGAGAGATGTGTAGTAAGAGACAGGACTACHVGGGGTATCTAATCC-3′. PCR was performed using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Inc., Wilmington, MA, USA), and PCR products were analyzed using a Bioanalyzer DNA 1000 chip (Agilent Technologies, Inc., Santa Clara, CA, USA). PCR products were purified using an Agencourt AMPure XP 60 mL kit (Beckman Coulter, Inc., Brea, CA, USA). Then, a second PCR was performed to construct a sequencing library, following the Illumina 16S Metagenomic Sequencing Library Preparation Protocol. The PCR products were purified and subjected to amplicon sequencing. Sample sequencing was performed using Illumina MiSeq (Illumina, Inc., San Diego, CA, USA), and paired-end sequence data detected in each sample were stored in separate FASTA files for Read1 and Read2. Sequence data were analyzed using Qiime2 [12]. First, demultiplexing and denoising were performed to remove adapter sequences, linker sequences, and primer sequences.

2.6. Microbial Population Analysis

Before analysis by Qiime2, ASVs (amplicon sequence variants) were generated by filtering, denoising, chimera checking, and merging paired reads of the sequence data according to the DADA2 workflow [13]. The Silva dataset was used for the 16S rRNA taxonomy database [14,15], and the V3-V4 region of the 16S rRNA was cut from the full-length sequence to adjust the dataset for analysis. The alpha diversity of each bacterial microbiota and the beta diversity of the detected sites were examined.

2.7. Statistical Analysis

To examine the frequency of periodontitis in patients with aortic valve disease, a one-sample Z-test was used as a statistical test to compare the proportion of stage III + IV + edentulous patients with aortic valve disease with the proportion of stage III + IV + edentulous patients in the general population (63.5%), adjusted for age based on the results of the 2016 Survey of Dental Diseases of Japan. Standard errors were calculated based on the population proportions, and one-tailed tests were performed. The significance level was set at 0.05. Statistical analyses were conducted using R version 4.2.3 (R Foundation for Statistical Computing, Vienna, Austria) [16]. We examined the stages of periodontitis and antibody titers against periodontal pathogenic bacteria using the Kruskal–Wallis test. The correlation between the antibody titer and the stage of periodontitis was analyzed.
The alpha diversity of each bacterial microbiota and the beta diversity of the detected sites were examined. To assess the alpha diversity, Pielou’s evenness index, reflecting the evenness of taxonomic distribution rather than phylogenetic breadth, and Shannon’s diversity index, which accounts for both species richness and evenness, were calculated. A Kruskal–Wallis test was used to compare the distributions among plaque, tongue, and valve groups [17]. A significance level of α = 0.05 was used. The analysis was conducted using the diversity alpha plugin in Qiime2 (version 2021.11) and compared between groups by the diversity alpha-group-significance plugin.
To analyze the beta diversity, principal coordinate analysis (PCoA) was performed based on the distance and read count values in the phylogenetic tree of the detected bacteria [18,19]. We used PERMANOVA (permutational multivariate analysis of variance) to test for significant differences in microbial community composition among groups, based on the unweighted UniFrac distance matrix [20]. The analysis was conducted using the ‘adonis2′ function from the ‘vegan’ R package (version 2.6.4) [21], with 999 permutations.

3. Results

3.1. Periodontitis and Aortic Valve Condition in Patients with Aortic Valve Disease

Thirty-two patients with AS or AR who underwent aortic valve replacement between May 2020 and March 2021 at Dokkyo Medical University Hospital were enrolled in this study. The patients’ demographic data are presented in Table 1. These patients underwent an oral examination and oral management prior to surgery, and their periodontal disease was staged based on an intraoral examination, panoramic radiograph, and periodontal pocket depth [9].
Of the 32 patients, 15 were males and 17 were females, with an average age of 74.15 ± 8.172 years (Table 1). Five patients had periodontal disease stage I, two patients had stage II, five had stage III, ten patients had stage IV, and ten patients were edentulous. Eleven patients had more than 21 teeth, six patients had 11 to 20 teeth, and five patients had 1 to 10 teeth. Twenty-six patients had calcification in the aortic valves, and seven were suspected to have bicuspid valves based on transthoracic echocardiography. Twenty-five patients had severe AS, two had moderate to severe AS, three had moderate AS, one had severe AR, and one had moderate to severe AR. Bacterial DNA was detected in 12 patients (37.5%) from resected aortic valves by PCR in a total of 32 patients examined. Sufficient bacterial DNA was collected in six patients (50%) from the aortic valves for metagenomic analysis in the twelve patients examined. ASVs in the V3–V4 region of the 16S rRNA in all six patients (100%) matched between oral bacteria and bacteria collected from the aortic valves. Detailed data for individual patients are presented in Table S1. AVAi averaged 0.45 ± 0.0012, SV averaged 78.93 ± 78.60, and SVi averaged 49.23 ± 27.57 (Table S1).
We compared the severity of periodontitis in the patients in this study and of the same age group from the results of the Survey of Actual Conditions of Dental Diseases in Japan [22]; 36.5% of the patients were classified as having mild disease in stages I and II, while 25.9% of patients with aortic disease were in the same category. In contrast, the percentage of patients with advanced periodontitis in stages III and IV and the edentulous jaw group was 74.1% in patients with aortic valve disease and 63.5% in the Survey of Dental Diseases in Japan (Figure 1). Statistical comparisons were difficult because there was a large difference in population size (32 patients in this study and 2378 patients in the survey), and 32 patients were not extracted from 2378 patients. Although this is an exploratory analysis rather than a causal analysis, there was a tendency for more patients with aortic valve disease to have stage III or IV advanced periodontitis and edentulous jaws (p = 0.042, Z-test).

3.2. Measurement of Serum Antibody Titer Against Periodontal Pathogenic Bacteria in Patients with Aortic Valve Disease

Periodontal pathogenic bacteria are classified into several categories (color-coded as red, orange, yellow, green, blue, and purple complexes) based on their importance in clinical pathogenesis by Socransky et al. [23], and bacteria classified in the red complex (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) are considered to be important for the pathogenesis of periodontitis (Figure 2A). The radar chart in Figure 2B shows the mean antibody titers of various periodontal pathogenic bacteria in the serum of patients with aortic valve disease. Antibody titers against bacteria in the red complex category were markedly elevated in patients compared to healthy controls. A comparison of serum antibody titers against red complex bacteria by periodontitis stage in patients with aortic valve disease showed that antibody titers tended to increase with the progression of the periodontitis stage, and antibody titers to red-complex bacteria were positively correlated with the periodontitis stage (Figure 2C). A weak but positive correlation was also observed between the antibody titer against the bacteria of the orange complex and the periodontitis stage (Figure 2C). Antibody titers against the bacteria of the blue and green complexes were weakly correlated with the periodontitis stage, although there was a tendency for antibody titers to increase.

3.3. Taxonomy Analysis

Bacterial 16S rRNA was detected in 12 aortic valves of 32 patients examined using PCR. We obtained sufficient DNA for amplicon sequencing analysis for six patients. Taxonomic analysis was performed to identify ASVs with >70% homology. The bacterial microbiota composition based on the read count of each ASV is shown in Figures S1 and S2. Surprisingly, several bacterial species were detected in the resected aortic valve. As expected, the bacterial microbiota of the tongue and periodontal pocket dental plaque were similar in many cases; however, the bacterial microbiota detected in the aortic valve showed a different pattern. Proteobacteria, Fusobacteriota, Firmicutes, Bacteroidota, and Actinobacteriota were found as the bacterial phyla with high read counts from the aortic valve (Figure S3). Some of the phyla were detected at similar frequencies at different sites in one patient, while others were detected at different frequencies in the aortic valve. In the present study, we did not search for individuals without periodontal disease or without aortic valve disease, so we were unable to verify whether or not oral dysbiosis occurs in patients with aortic valve disease by means of bacterial phyla detected on the tongue or in periodontal pockets. Furthermore, the organs in which the bacterial phyla detected in the aortic valve are endemic are diverse, and although many bacteria are endemic in the oral cavity, we cannot conclude that the oral cavity is a gateway for bacterial entry based on this alone. The same results were obtained, excluding the cases in which the second round PCR was carried out when performing metagenomic analysis. In general, the experiment using PCR carried the risk of sample cross-contamination, but the results of the analysis indicated that no cross-contamination of samples occurred.

3.4. α-Diversity and β-Diversity

Examination of α-diversity and β-diversity indicated that the bacterial microbiota in the resected aortic valve tended to be similar, although not identical to the composition of the oral bacterial microbiota. We examined the α-diversity of the plaques, tongue swabs, and excised aortic valves (Figure S2). There was no significant difference in the diversity within each bacterial microbiota detected at each site (p = 0.547). This suggests that a wide variety of bacteria are present in the resected aortic valve, and that, like the oral environment, the bacterial microbiota may be diverse. A Kruskal–Wallis test against Pielou’s evenness index revealed no significant difference among the three groups, H = 1.205, p = 0.547. Post hoc analysis using Bonferroni correction also showed no difference between groups (Figure S4A, Table S2). A Kruskal–Wallis test against Shanon’s index revealed no significant difference among the three groups, H = 0.889, p = 0.641. Post hoc analysis using Bonferroni correction also showed no difference between groups (Figure S4B, Table S3). When we examined the β-diversity among the bacterial microbiota (Figure S5), we found that the vectors of diversity of the bacterial microbiota differed among the three groups (p = 0.001): plaque, tongue, and resected aortic valve. Among the resected aortic valves, we recognized two groups with different vectors of diversity, and among the two groups, three cases, AS 14, 20, and 25, were considered to have bacterial microbiota similar to those of the oral-derived specimens, being closer to the oral bacteria. The frequency of detection of ASVs in AS 18, 28, and 30 was higher than that in the Unassigned group, which may have been a factor in the division into two groups. Although the Gene Analyzer detected the expected size of PCR products in these three samples at the first PCR stage, it was insufficient in quantity. As the primary purpose of this study was to examine whether bacteria were present in the resected aortic valve, we performed the first PCR again using the first PCR product as a PCR template. This increased nonspecific amplification, and unassigned ASVs became more frequent. However, from the viewpoint of Axes 2 and 3, there was a similar trend to that of the oral-derived specimens. A significant difference in microbial composition was observed among the three groups (PERMANOVA: F = 3.025, R2 = 0.287, p = 0.002) (Table S4).

3.5. Characteristics of Bacteria Detected in the Tongue, Dental Plaque, and Resected Aortic Valve

The 195 bacteria detected in the tongue, dental plaque, and resected aortic valve specimens were ranked according to read count (Figures S1–S3 and S6). The lists of the top 25 bacteria are shown in Figure S6A–C. Among the bacteria detected in the resected aortic valve, oral commensal bacteria and periodontal pathogenic bacteria (color-coded as red, orange, yellow, green, blue, and purple) according to Socransky’s classification [23] were extracted and are shown in Figure 3. Among the bacteria listed for resected aortic valves, 48% (16–64%) of the 35 species were associated with the oral cavity, and 18% (4–28%) were periodontal pathogenic bacteria (Figure S6A–C). In cases 20 and 25, red and orange bacteria were detected in the aortic valve. In case 30, neither red nor orange complex bacteria were detected in the aortic valve (Figure 3). In cases 14, 18, and 28, red and orange bacteria were detected in the aortic valve (Figure 3), although the bacterial microbiota detected on the dorsal surface of the tongue contained no red and only a few orange bacteria (Figure S6C). In cases 20, 25, and 30, a significant number of red and orange bacteria were included in the bacterial microbiota considered endemic to the oral cavity detected on the dorsal surface of the tongue (Figure S6B,C).

3.6. Identification of the Same ASV in the V3 -V4 Region of the 16S rRNA Sequence in the Tongue, Dental Plaque, and Resected Aortic Valve

The same ASV in the V3 -V4 region of the 16S rRNA sequence was detected in the tongue, dental plaque, and the resected aortic valve (Figure 4). This indicates that bacteria with the same genetic background, that is, strain-level similarity of the bacteria, are shown in the oral cavity and aortic valve. A total of 2524 independent ASVs were detected in all samples. Forty bacteria were found to have identical ASVs in the excised aortic valve and oral cavity (dorsal surface of the tongue and/or dental plaque). Fourteen bacteria were detected with identical ASVs from the three sites of the tongue, dental plaque, and excised aortic valve, and twenty-six bacteria were detected with identical ASVs from the excised aortic valve and tongue or dental plaque (Figure 4). Twelve of the forty bacteria were periodontal pathogenic bacteria, according to Socransky’s classification. Although the possibility that the bacteria detected from the aortic valve were transient adherences of oral bacteria due to bacteremia cannot be completely ruled out, it is considered extremely unlikely because of the intravenous administration of antibiotics during the perioperative period, the physical oscillation during the resection, and the saline rinse of the resected valve.

4. Discussion

In this study, we found several novel points: (1) patients with aortic valve disease were more likely to have periodontitis and more of them had severe disease or edentulous jaws; (2) serum titers of antibodies against periodontal pathogenic bacteria and red complexes were high in patients with aortic valve disease; (3) bacterial DNA was detectable in the resected aortic valve of 12/32 (37.5%) patients with aortic valve disease; (4) a wide variety of bacteria were detected in the resected aortic valves of six patients who underwent amplicon sequencing analysis; (5) 40 bacteria with strain-level similarity were found in both the resected aortic valve and oral bacterial ASVs; (6) among the 40 bacteria with strain-level similarity, 12 were periodontal pathogenic bacteria according to Socransky’s classification. These results strongly suggest that in patients with current periodontitis or previous periodontitis, oral bacteria continuously invade the bloodstream and settle in the aortic valve.
A potential limitation of this study is whether the fact that bacterial DNA was detected in the aortic valve in only 12 of 32 cases, and that sufficient bacterial DNA was recovered for metagenomic analysis in only 6 cases, is a result of manipulation or a genuine finding. In the future, it will be necessary to quantify the percentage of patients who have the same clone of oral bacteria in the aortic valve and to strictly control the detection limit of bacterial DNA from the valve to prevent cross-contamination. As this study is a PCR-based experiment, increasing the sensitivity reduces the specificity. Increasing the number of patients and setting a strict detection sensitivity threshold makes it possible to determine whether the detection frequency obtained in this experiment reflects the true situation or is merely a reflection of the detection sensitivity. Furthermore, since we are unable to examine the presence of oral bacteria in the aortic valves of healthy subjects or in patients with mild aortic valve disease, it is not known whether this observation is a phenomenon specific to patients with severe aortic valve disease. A study investigating the presence of bacteria in heart samples taken during autopsies of individuals without aortic valve disease who died in accidents could confirm whether the findings of this study are specific to patients with aortic valve disease. In AS 18, 28, and 30, samples at the first PCR stage were insufficient in quantity to construct a metagenome library. As the primary purpose of this study was to examine whether oral bacteria were present in the resected aortic valve, we performed PCR again using the first PCR product as a template. This increased nonspecific amplification, and unassigned ASVs became more frequent. However, examining the ASVs identified in these three patients reveals no evidence of contamination among the samples. In these three patients, the bacteria detected in the aortic valve included the same ASV as the oral bacteria, proving that they were bacterial with strain-level similarity in each patient.
In some cases (cases 20, 25, and 30), bacteria in the red and orange complexes were increased in the bacterial microbiota of the oral cavity. It was endemic to the oral cavity and was detected on the dorsal surface of the tongue, suggesting that dysbiosis had occurred. In cases 20 and 25, bacteria in the red and orange complexes were also detected in the aortic valve. In contrast, the dorsal surface of the tongue contained fewer orange complex bacteria in cases 14, 18, and 28, without red complexes. Nevertheless, red and orange complexes were detected in the aortic valve, suggesting that colonization of the aortic valve via bacteremia from periodontal bacteria in the periodontal pocket occurs even in the absence of oral bacterial dysbiosis. The similarity of the bacteria detected in the oral cavity (dorsal surface of the tongue or periodontal pockets) and the aortic valve was examined. In cases 14, 18, 20, and 25, a significant number of bacteria were detected from both sources. Only Staphylococcus and Neisseria were detected in cases 28 and 30, respectively, suggesting that oral bacteria may play a lesser role.
In many cases, Streptococcus, which appears to be an early colonizer and a subsequent dental plaque constituent bacteria, was also present in the aortic valve, suggesting that they may be involved in bacterial colonization. However, some ASV species that were detected in the resected aortic valve were not detected in the oral cavity. Because oral dysbiosis has been present for a considerable period, and the timing of bacterial invasion and settlement in the aortic valve does not always coincide due to repeated acute conversion and remission of periodontal disease, it is thought that there were bacteria that existed in the oral cavity but whose ASVs did not align.
Although numerous reports have documented the detection of oral bacteria in distant organs using PCR [8,24,25,26,27,28,29,30,31,32,33], few studies have confirmed whether the bacteria detected in the oral cavity and distant organs are the same clone [8]. For example, Zeibolz et al. examined the presence of pathogenic periodontal bacteria in aortic valves using PCR [24]. The relationship between oral bacteria and diseases of distant organs is also well known. Periodontal pathogens are known to be risk factors for atherosclerosis [25,26,27,28,29] and, as a result, are thought to be involved in the development of several cardiac diseases, including infective endocarditis [30]. Streptococcus mutans, a well-known bacterium that causes caries, is also involved in the development of cerebral hemorrhage [31,32,33]. Recent studies have demonstrated that oral bacteria can enter the bloodstream and colonize heart valves and blood vessels [34,35,36,37,38]. This may lead to chronic low-grade infections and promote inflammation and calcification in the vascular walls, potentially contributing to the development and progression of cardiovascular diseases. Although these findings are valuable as circumstantial evidence that oral bacteria can infect distant organs via the bloodstream, it is challenging to determine whether diseases in the oral cavity, such as periodontitis and dental caries, can serve as a gateway for entry. In this study, we found that bacteria with strain-level similarity, possibly the same clones, were present in the oral cavity, distant organs, and the aortic valve. However, at present, we have not clarified whether oral bacteria caused aortic valve disease or colonized the injured aortic valve.
The importance of oral management during surgery and several medical treatments has recently been reported [39]. However, there is no clear direct evidence that bacteria in the oral cavity can induce secondary infections in other organs. Accumulated experience strongly suggests that oral lesions (dental caries reaching the root canal or periodontal disease) may serve as a gateway for bacterial invasion into the body, and studies providing direct evidence have increased. In this study, we detected bacteria with strain-level similarity, that is, possibly the same clone of bacteria, in the oral cavities and resected aortic valves of patients with aortic valve disease. Oral dysbiosis and the resulting potential bacteremia are associated with the pathogenesis of aortic valve diseases.

5. Conclusions

Oral dysbiosis and the resulting potential bacteremia are associated with the pathogenesis of aortic valve diseases. Appropriate oral management may contribute to the prevention of aortic valve diseases. Further experiments may be necessary to demonstrate the direct association between oral bacteria and the onset or progression of aortic valve disease.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms13071677/s1, Figure S1: Composition of the bacterial microbiota at the genus level in samples of dissected aortic valve, dental plaque, and tongue swabs; Figure S2: Heat map of the bacterial microbiota at the genus level in samples of dissected aortic valve, dental plaque, and tongue swabs; Figure S3: Heat map of the bacterial microbiota at the phylum level in samples of dissected aortic valve, dental plaque, and tongue swabs; Figure S4: Comparison of alpha diversity for each sample group; Figure S5: Principal coordinates analysis of each sample to examine beta diversity among groups.; Figure S6: Top 25 bacteria listed by read count in each sample; Table S1: Detailed demographic data of the AS/AR patient; Table S2: Statistical analysis of Pielou’s evenness index among dental plaque, tongue swab, and dissected aortic valve groups; Table S3: Statistical analysis of Shannon’s diversity index among dental plaque, tongue swab, and dissected aortic valve groups; Table S4: PERMANOVA results based on unweighted UniFrac distances among dental plaque, tongue swab, and dissected aortic valve.

Author Contributions

Conceptualization: Y.K., E.Y. and H.K.; Methodology: Y.K., E.Y. and H.K.; Conceptualization: Y.K., E.Y. and H.K.; Methodology: Y.K., E.Y. and H.K.; Investigation: E.Y., Y.K., R.S., T.H., S.I. (Shu Inami), I.S. and T.S.; Visualization: Y.K., E.Y., S.I. (Shu Inami) and T.S.; Funding acquisition: E.Y. and Y.K.; Project administration: Y.K. and E.Y.; Supervision: H.S., S.H., H.F., S.T., C.F., S.I. (Sayaka Izumi), T.W. and H.K.; Writing—original draft: Y.K. and E.Y.; Writing—review and editing: Y.K., E.Y. and H.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by JSPS KAKENHI Grant Number JP20K10123 (Y.K.), Dokkyo Medical University Grant in Aid No. 1864 (E.Y.), and Dokkyo Medical University Grant in Aid No. 2153 (E.Y.).

Institutional Review Board Statement

This study was conducted in accordance with the Declaration of Helsinki and the Ethical Guidelines for Medical and Health Research Involving Human Subjects issued by the Japan Ministry of Health, Labor and Welfare and approved by the Ethics Committee of Dokkyo Medical University (Approval as No. R-16-14J on 11 November 2018 and No. R-37-20J on 12 October 2021).

Informed Consent Statement

Written informed consent was obtained from all participants. In cases where the patients themselves could not decide, we explained the details of this research to the legal guardian of the patient. Written informed consent has been obtained from the patients to publish this study.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Acknowledgments

We would like to thank all the staff members of the Department of Oral and Maxillofacial Surgery and the Bacteriology Laboratory of Dokkyo Medical University Hospital for their useful advice and support regarding sample collection from the oral cavity. We also appreciate all the staff members of the operating room of Dokkyo Medical University Hospital for their support with sample collection. We are grateful to Yuki Hirano and Mayumi Sakata for coordinating the schedule and liaison for collecting the surgical specimens.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of this study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

AS: aortic stenosis. AR: aortic regurgitation. AVA: aortic valve area. AVAi: AVA index. LVEF: left ventricular ejection fraction. peakPG: peak pressure gradient. SV: stroke volume. SVi: stroke volume index. MR: mitral regurgitation. TR: tricuspid regurgitation. PR: pulmonary regurgitation. PH: pulmonary hypertension. OMI: old myocardial infarction. PMI: pacemaker implantation. TAVI: transcatheter aortic valve implantation. NA: not applicable. ND: not determined. In valvular findings: -, no sign of regurgitation; ±, trace level of regurgitation; +, mild regurgitation; ++, moderate regurgitation; +++, severe regurgitation.

References

  1. Kumar, P.S. From focal sepsis to periodontal medicine: A century of exploring the role of the oral microbiome in systemic disease. J. Physiol. 2017, 595, 465–476. [Google Scholar] [CrossRef] [PubMed]
  2. Freire, K.; Nelson, K.E.; Edlund, A. The oral host–microbial interactome: An ecological chronometer of health? Trends Microbiol. 2021, 29, 551–561. [Google Scholar] [CrossRef] [PubMed]
  3. Sampaio-Maia, B.; Caldas, I.M.; Pereira, M.L.; Pérez-Mongiovi, D.; Araujo, R. The oral microbiome in health and its implication in oral and systemic diseases. Adv. Appl. Microbiol. 2016, 97, 171–210. [Google Scholar] [CrossRef] [PubMed]
  4. Yoshida, S.; Nakagawa, H.; Kuwata, H.; Nabuchi, A.; Yaso, A.; Shirota, T. Metagenomic analysis of oral plaques and aortic valve tissues reveals oral bacteria associated with aortic stenosis. Clin. Oral Investig. 2023, 27, 4335–4344. [Google Scholar] [CrossRef] [PubMed]
  5. Pardo, A.; Signoriello, A.; Signoretto, C.; Messina, E.; Carelli, M.; Tessari, M.; De Manna, N.D.; Rossetti, C.; Albanese, M.; Lombardo, G. Detection of periodontal pathogens in oral samples and cardiac specimens in patients undergoing aortic valve replacement: A pilot study. J. Clin. Med. 2021, 10, 3874. [Google Scholar] [CrossRef] [PubMed]
  6. Joseph, J.; Naqvi, S.Y.; Giri, J.; Goldberg, S. Aortic stenosis: Pathophysiology, diagnosis, and therapy. Am. J. Med. 2017, 130, 253–263. [Google Scholar] [CrossRef] [PubMed]
  7. Boskovski, M.T.; Gleason, T.G. Current therapeutic options in aortic stenosis. Circ. Res. 2021, 128, 1398–1417. [Google Scholar] [CrossRef] [PubMed]
  8. Hakata, K.; Kawamata, H.; Imai, Y. Implication of the oral bacteria on the onset of infective endocarditis. Dokkyo J. Med. Sci. 2014, 41, 103–113. [Google Scholar] [CrossRef]
  9. Caton, J.G.; Armitage, G.; Berglundh, T.; Chapple, I.L.C.; Jepsen, S.; Kornman, K.S.; Mealey, B.L.; Papapanou, P.N.; Sanz, M.; Tonetti, M.S. A new classification scheme for periodontal and peri-implant diseases and conditions–introduction and key changes from the 1999 classification. J. Clin. Periodontol. 2018, 45 (Suppl. 20), S1–S8. [Google Scholar] [CrossRef] [PubMed]
  10. Nishi, H.; Hosomi, N.; Ohta, K.; Aoki, S.; Nakamori, M.; Nezu, T.; Shigeishi, H.; Shintani, T.; Obayashi, T.; Ishikawa, K.; et al. Serum immunoglobulin G antibody titer to Fusobacterium nucleatum is associated with unfavorable outcome after stroke. Clin. Exp. Immunol. 2020, 200, 302–309. [Google Scholar] [CrossRef] [PubMed]
  11. Aydin, S. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides 2015, 72, 4–15. [Google Scholar] [CrossRef] [PubMed]
  12. Bolyen, E.; Rideout, J.R.; Dillon, M.R.; Bokulich, N.A.; Abnet, C.C.; Al-Ghalith, G.A.; Alexander, H.; Alm, E.J.; Arumugam, M.; Asnicar, F.; et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 2019, 37, 852–857. [Google Scholar] [CrossRef] [PubMed]
  13. Callahan, B.J.; McMurdie, P.J.; Rosen, M.J.; Han, A.W.; Johnson, A.J.; Holmes, S.P. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 2016, 13, 581–583. [Google Scholar] [CrossRef] [PubMed]
  14. Quast, C.; Pruesse, E.; Yilmaz, P.; Gerken, J.; Schweer, T.; Yarza, P.; Peplies, J.; Glöckner, F.O. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 2013, 41, D590–D596. [Google Scholar] [CrossRef] [PubMed]
  15. Robeson, M.S.; O’Rourke, D.R.; Kaehler, B.D.; Ziemski, M.; Dillon, M.R.; Foster, J.T.; Bokulich, N.A. RESCRIPt: Reproducible sequence taxonomy reference database management. PLoS Comput. Biol. 2021, 17, e1009581. [Google Scholar] [CrossRef] [PubMed]
  16. R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2023; Available online: https://www.R-project.org/ (accessed on 3 March 2022).
  17. Fan, C.; Zhang, D. A note on power and sample size calculations for the Kruskal-Wallis test for ordered categorical data. J Biopharm. Stat. 2012, 22, 1162–1173. [Google Scholar] [CrossRef] [PubMed]
  18. Lozupone, C.A.; Knight, R. Species divergence and the measurement of microbial diversity. FEMS Microbiol. Rev. 2008, 32, 557–578. [Google Scholar] [CrossRef] [PubMed]
  19. Wang, Y.; Sun, F.; Lin, W.; Zhang, S. AC-PCoA: Adjustment for confounding factors using principal coordinate analysis. PLoS Comput. Biol. 2022, 18, e1010184. [Google Scholar] [CrossRef] [PubMed]
  20. Yue, Y.; Hu, Y.J. Extension of PERMANOVA to Testing the Mediation Effect of the Microbiome. Genes 2022, 13, 940. [Google Scholar] [CrossRef] [PubMed]
  21. Oksanen, J.; Simpson, G.; Blanchet, F.; Kindt, R.; Legendre, P.; Minchin, P.; O’Hara, R.; Solymos, P.; Stevens, M.; Szoecs, E.; et al. Vegan: Community Ecology Package, R package version 2.6-4; 2022. Available online: https://cran.r-project.org/src/contrib/Archive/vegan/ (accessed on 26 June 2025).
  22. Japanese Ministry of Health, Labour and Welfare. Heisei 28 Nen Shikasikkanjittaityousa [Dental Disease Survey]; Japanese Ministry of Health, Labour and Welfare: Tokyo, Japan, 2016.
  23. Socransky, S.; Haffajee, A. Dental biofilms: Difficult therapeutic targets. Periodontol. 2000 2002, 28, 12–55. [Google Scholar] [CrossRef] [PubMed]
  24. Ziebolz, D.; Jahn, C.; Pegel, J.; Semper-Pinnecke, E.; Mausberg, R.F.; Waldmann-Beushausen, R.; Schöndube, F.A.; Danner, B.C. Periodontal bacteria DNA findings in human cardiac tissue—Is there a link of periodontitis to heart valve disease? Int. J. Cardiol. 2018, 251, 74–79. [Google Scholar] [CrossRef] [PubMed]
  25. Bartova, J.; Sommerova, P.; Lyuya-Mi, Y.; Mysak, J.; Prochazkova, J.; Duskova, J.; Janatova, T.; Podzimek, S. Periodontitis as a risk factor of atherosclerosis. J. Immunol. Res. 2014, 2014, 636893. [Google Scholar] [CrossRef] [PubMed]
  26. Olsen, I.; Taubman, M.A.; Singhrao, S.K. Porphyromonas gingivalis suppresses adaptive immunity in periodontitis, atherosclerosis, and Alzheimer’s disease. J. Oral Microbiol. 2016, 8, 33029. [Google Scholar] [CrossRef] [PubMed]
  27. Schenkein, H.A.; Papapanou, P.N.; Genco, R.; Sanz, M. Mechanisms underlying the association between periodontitis and atherosclerotic disease. Periodontol. 2000 2020, 83, 90–106. [Google Scholar] [CrossRef] [PubMed]
  28. Fernandes, C.P.; Oliveira, F.A.F.; de Barros Silva, P.G.; Alves, A.P.N.N.; Mota, M.R.L.; Montenegro, R.C.; Burbano, R.M.R.; Seabra, A.D.; Filho, J.G.L.; Lima, D.L.F.; et al. Molecular analysis of oral bacteria in dental biofilm and atherosclerotic plaques of patients with vascular disease. Int. J Cardiol. 2014, 174, 710–712. [Google Scholar] [CrossRef] [PubMed]
  29. Rath, S.K.; Mukherjee, M.; Kaushik, R.; Sen, S.; Kumar, M. Periodontal pathogens in atheromatous plaque. Indian J. Pathol. Microbiol. 2014, 57, 259–264. [Google Scholar] [CrossRef] [PubMed]
  30. Carrizales-Sepúlveda, E.F.; Ordaz-Farías, A.; Vera-Pineda, R.; Flores-Ramírez, R. Periodontal disease, systemic inflammation and the risk of cardiovascular disease. Heart Lung Circ. 2018, 27, 1327–1334. [Google Scholar] [CrossRef] [PubMed]
  31. Pyysalo, M.J.; Pyysalo, L.M.; Pessi, T.; Karhunen, P.J.; Öhman, J.E. The connection between ruptured cerebral aneurysms and odontogenic bacteria. J. Neurol. Neurosurg. Psychiatry 2013, 84, 1214–1218. [Google Scholar] [CrossRef] [PubMed]
  32. Taniguchi, Y.; Ouhara, K.; Kitagawa, M.; Akutagawa, K.; Kawada-Matsuo, M.; Tamura, T.; Zhai, R.; Hamamoto, Y.; Kajiya, M.; Matsuda, S.; et al. Periapical lesion following Cnm-positive Streptococcus mutans pulp infection worsens cerebral hemorrhage onset in an SHRSP rat model. Clin. Exp. Immunol. 2022, 210, 321–330. [Google Scholar] [CrossRef] [PubMed]
  33. Hosoki, S.; Saito, S.; Tonomura, S.; Ishiyama, H.; Yoshimoto, T.; Ikeda, S.; Ikenouchi, H.; Yamamoto, Y.; Hattori, Y.; Miwa, K.; et al. Oral carriage of Streptococcus mutans harboring the cnm gene relates to an increased incidence of cerebral microbleeds. Stroke 2020, 51, 3632–3639. [Google Scholar] [CrossRef] [PubMed]
  34. Oliveira, F.A.F.; Forte, C.P.F.; de Barros Silva, P.G.; Lopes, C.B.; Montenegro, R.C.; Dos Santos, Â.K.C.R.; Sobrinho, C.R.M.R.; Mota, M.R.L.; Sousa, F.B.; Alves, A.P.N.N. Molecular Analysis of Oral Bacteria in Heart Valve of Patients With Cardiovascular Disease by Real-Time Polymerase Chain Reaction. Medicine 2015, 94, e2067. [Google Scholar] [CrossRef] [PubMed]
  35. Nakano, K.; Nemoto, H.; Nomura, R.; Inaba, H.; Yoshioka, H.; Taniguchi, K.; Amano, A.; Ooshima, T. Detection of oral bacteria in cardiovascular specimens. Oral Microbiol. Immunol. 2009, 24, 64–68. [Google Scholar] [CrossRef] [PubMed]
  36. Cohen, D.J.; Malave, D.; Ghidoni, J.J.; Iakovidis, P.; Everett, M.M.; You, S.; Liu, Y.; Boyan, B.D. Role of oral bacterial flora in calcific aortic stenosis: An animal model. Ann. Thorac. Surg. 2004, 77, 537–543. [Google Scholar] [CrossRef] [PubMed]
  37. Raffaelli, L.; Santangelo, R.; Falchetti, P.; Galluccio, F.; Luciani, N.; Anselmi, A.; Nowzari, H.; Verdugo, F.; Fadda, G.; D’ADdona, A. Examination of periodontal pathogens in stenotic valve specimens and in whole blood samples in patients affected by aortic valve stenosis and chronic periodontitis. Int. J. Immunopathol. Pharmacol. 2010, 23, 561–566. [Google Scholar] [CrossRef] [PubMed]
  38. Hokamura, K.; Umemura, K. Roles of oral bacteria in cardiovascular diseases—From molecular mechanisms to clinical cases: Porphyromonas gingivalis is the important role of intimal hyperplasia in the aorta. J. Pharmacol. Sci. 2010, 113, 110–114. [Google Scholar] [CrossRef] [PubMed]
  39. Tsubura-Okubo, M.; Komiyama, Y.; Kamimura, R.; Sawatani, Y.; Arai, H.; Mitani, K.; Haruyama, Y.; Kobashi, G.; Ishihama, H.; Uchida, D.; et al. Oral management with polaprezinc solution reduces adverse events in hematopoietic stem cell transplantation patients. Int. J. Oral Maxillofac. Surg. 2021, 50, 906–914. [Google Scholar] [CrossRef] [PubMed]
Microorganisms 13 01677 g001
Figure 1. Periodontitis/edentulous status of patients with aortic valve disease. Stage represents the severity of periodontitis. Stages I and II were considered as early/mild periodontitis, while stages III and IV were considered as advanced and moderate to severe periodontitis. The edentulous patient was considered a result of periodontitis progression. There was a tendency for more patients with aortic valve disease to have stages III or IV advanced periodontitis and edentulous jaws (Z = 1.72, p = 0.042, one-tailed test). Bars indicate 95% confidence intervals. *: p = 0.042.
Figure 1. Periodontitis/edentulous status of patients with aortic valve disease. Stage represents the severity of periodontitis. Stages I and II were considered as early/mild periodontitis, while stages III and IV were considered as advanced and moderate to severe periodontitis. The edentulous patient was considered a result of periodontitis progression. There was a tendency for more patients with aortic valve disease to have stages III or IV advanced periodontitis and edentulous jaws (Z = 1.72, p = 0.042, one-tailed test). Bars indicate 95% confidence intervals. *: p = 0.042.
Microorganisms 13 01677 g001
Microorganisms 13 01677 g002aMicroorganisms 13 01677 g002b
Figure 2. Status of antibody titer to periodontal pathogenic bacteria. (A) Schematic illustration of periodontal pathogenic bacteria classified by Socransky’s criteria. Periodontal pathogenic bacteria exist in a defined order. They are divided into six categories, and each is coded by color. Blue, purple, green, and yellow complexes form the basic attachment to the body surfaces such as teeth and mucosa. The red complex is a definite pathogen in periodontitis, while the orange complex primarily functions as a connecting anchor for the members of the red complex within the biofilm. (B) Average antibody titer against representative periodontal pathogenic bacteria. Antibody titers against each pathogenic bacterium are standardized to the average value of healthy controls. The red area in the radar chart demonstrates a +3 S.D. titer from healthy controls, the orange area a +2 S.D., and the green area a +1 S.D., respectively. The average of AS/AR patients in this study is shown in the red line. Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tannerella forsythia (Tf), Aggregatibacter actinomycetemcomitans (Aa), Eikenella corrodens (Ec), Fusobacterium nucleatum (Fn), Prevotella intermedia (Pi), Prevotella nigrescens (Pn), and Campylobacter rectus (Cr). (C) Correlation of antibody titers against the periodontal pathogenic bacteria and periodontal stage of AS/AR patient. Antibody titers against bacteria classified into red, orange, blue, and green complexes were summed as an index and plotted for the 13 patients who had remaining teeth and for whom serum could be collected. The antibody titer of healthy individuals was set to 100. Lines in the figure demonstrate regression lines.
Figure 2. Status of antibody titer to periodontal pathogenic bacteria. (A) Schematic illustration of periodontal pathogenic bacteria classified by Socransky’s criteria. Periodontal pathogenic bacteria exist in a defined order. They are divided into six categories, and each is coded by color. Blue, purple, green, and yellow complexes form the basic attachment to the body surfaces such as teeth and mucosa. The red complex is a definite pathogen in periodontitis, while the orange complex primarily functions as a connecting anchor for the members of the red complex within the biofilm. (B) Average antibody titer against representative periodontal pathogenic bacteria. Antibody titers against each pathogenic bacterium are standardized to the average value of healthy controls. The red area in the radar chart demonstrates a +3 S.D. titer from healthy controls, the orange area a +2 S.D., and the green area a +1 S.D., respectively. The average of AS/AR patients in this study is shown in the red line. Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tannerella forsythia (Tf), Aggregatibacter actinomycetemcomitans (Aa), Eikenella corrodens (Ec), Fusobacterium nucleatum (Fn), Prevotella intermedia (Pi), Prevotella nigrescens (Pn), and Campylobacter rectus (Cr). (C) Correlation of antibody titers against the periodontal pathogenic bacteria and periodontal stage of AS/AR patient. Antibody titers against bacteria classified into red, orange, blue, and green complexes were summed as an index and plotted for the 13 patients who had remaining teeth and for whom serum could be collected. The antibody titer of healthy individuals was set to 100. Lines in the figure demonstrate regression lines.
Microorganisms 13 01677 g002aMicroorganisms 13 01677 g002b
Microorganisms 13 01677 g003
Figure 3. Oral commensal bacteria and periodontal pathogenic bacteria detected in the resected aortic valve. Among the bacteria detected in the resected aortic valve, oral commensal bacteria and periodontal pathogenic bacteria (color-coded as red, orange, yellow, green, blue, and purple), according to Socransky’s classification, were extracted from the lists of the top 25 bacteria (Figure S6A) and are shown in Figure 3. In cases 20 and 25, red and orange bacteria were detected in the aortic valve. In case 30, neither red nor orange complex bacteria were detected in the aortic valve. In cases 14, 18, and 28, red and orange bacteria were detected in the aortic valve.
Figure 3. Oral commensal bacteria and periodontal pathogenic bacteria detected in the resected aortic valve. Among the bacteria detected in the resected aortic valve, oral commensal bacteria and periodontal pathogenic bacteria (color-coded as red, orange, yellow, green, blue, and purple), according to Socransky’s classification, were extracted from the lists of the top 25 bacteria (Figure S6A) and are shown in Figure 3. In cases 20 and 25, red and orange bacteria were detected in the aortic valve. In case 30, neither red nor orange complex bacteria were detected in the aortic valve. In cases 14, 18, and 28, red and orange bacteria were detected in the aortic valve.
Microorganisms 13 01677 g003
Microorganisms 13 01677 g004
Figure 4. List of bacteria to which the ASV was matched in dental plaque, tongue, and valve. Matched samples are highlighted by a red background. The numbers in the tables represent the read count of each bacterium. The color of the background of the bacteria name on the left column was the same color as the bacteria matching Sokransky’s color code in Figure 2. #: case number, P: dental plaque, T: dorsal surface of tongue, V: aortic valve.
Figure 4. List of bacteria to which the ASV was matched in dental plaque, tongue, and valve. Matched samples are highlighted by a red background. The numbers in the tables represent the read count of each bacterium. The color of the background of the bacteria name on the left column was the same color as the bacteria matching Sokransky’s color code in Figure 2. #: case number, P: dental plaque, T: dorsal surface of tongue, V: aortic valve.
Microorganisms 13 01677 g004
Table 1. Demographic data of the AS/AR patient.
Table 1. Demographic data of the AS/AR patient.
Age
Mean ± 1SD74.125 ± 8.172
Median76
N%
Gender
male1753.125
female1546.875
Stage of periodontitis
I515.625
II26.25
III515.625
IV1031.25
edentulous1031.25
Number of remaining teeth
21~1134.375
11~20618.75
1~10515.625
Valvular findings (duplicate)
calcification2681.25
bicuspid721.875
no abnormal findings412.5
Aortic valve disease
severe AS2578.125
moderate–severe AS26.25
moderate AS39.375
severe AR13.125
moderate–severe AR13.125
Number of patients with bacterial DNA detected from resected aortic valves by PCR; in total 32 patients examined1237.5
Number of patients with sufficient bacterial DNA collected from aortic valves for metagenomic analysis in the 12 patients examined650
Number of patients with 100% ASV match between oral bacteria and bacteria collected from aortic valves in the 6 patients examined6100
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Yaguchi, E.; Komiyama, Y.; Inami, S.; Shibasaki, I.; Shintani, T.; Shiraishi, R.; Hyodo, T.; Shiba, H.; Hamaguchi, S.; Fukuda, H.; et al. Oral Dysbiosis Is Associated with the Pathogenesis of Aortic Valve Diseases. Microorganisms 2025, 13, 1677. https://doi.org/10.3390/microorganisms13071677

AMA Style

Yaguchi E, Komiyama Y, Inami S, Shibasaki I, Shintani T, Shiraishi R, Hyodo T, Shiba H, Hamaguchi S, Fukuda H, et al. Oral Dysbiosis Is Associated with the Pathogenesis of Aortic Valve Diseases. Microorganisms. 2025; 13(7):1677. https://doi.org/10.3390/microorganisms13071677

Chicago/Turabian Style

Yaguchi, Erika, Yuske Komiyama, Shu Inami, Ikuko Shibasaki, Tomoaki Shintani, Ryo Shiraishi, Toshiki Hyodo, Hideki Shiba, Shinsuke Hamaguchi, Hirotsugu Fukuda, and et al. 2025. "Oral Dysbiosis Is Associated with the Pathogenesis of Aortic Valve Diseases" Microorganisms 13, no. 7: 1677. https://doi.org/10.3390/microorganisms13071677

APA Style

Yaguchi, E., Komiyama, Y., Inami, S., Shibasaki, I., Shintani, T., Shiraishi, R., Hyodo, T., Shiba, H., Hamaguchi, S., Fukuda, H., Toyoda, S., Fukumoto, C., Izumi, S., Wakui, T., & Kawamata, H. (2025). Oral Dysbiosis Is Associated with the Pathogenesis of Aortic Valve Diseases. Microorganisms, 13(7), 1677. https://doi.org/10.3390/microorganisms13071677

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop