Bacterial Infections

Contribution of Severe Dental Caries Induced by Streptococcus mutans to the Pathogenicity of Infective Endocarditis

Ryota Nomura, Saaya Matayoshi, Masatoshi Otsugu, Takahiro Kitamura, Noboru Teramoto, Kazuhiko Nakano
Nancy E. Freitag, Editor
DOI: 10.1128/IAI.00897-19

ABSTRACT

Streptococcus mutans, a major pathogen of dental caries, is regarded as a causative agent of infective endocarditis (IE), which mainly occurs in patients with underlying heart disease. However, it remains unknown whether severe dental caries that extend to pulp space represent a possible route of infection. In the present study, we evaluated the virulence of S. mutans for IE development using rats with concurrent severe dental caries and heart valve injury. Dental caries was induced in rats through the combination of a caries-inducing diet and the administration of S. mutans into the oral cavity. Then, the heart valves of a subset of rats were injured using a sterile catheter and wire under general anesthesia. The rats were euthanized at various times with various stages of dental caries. The number of teeth affected by dental caries with pulp exposure was increased in the rats in a time-dependent manner. S. mutans was recovered from injured heart tissue, which was mainly observed in rats with higher number of S. mutans bacteria in mandibular bone and a larger number of teeth in which caries extended to pulp. Dental caries was more severe in rats with heart injury than in rats without heart injury. Sequencing analysis targeting 16S rRNA revealed that specific oral bacteria appeared only in rats with heart injury, which may be related to the development of dental caries. Our findings suggest that dental caries caused by the combination of S. mutans infection and sucrose intake may contribute to S. mutans colonization in injured heart tissue.

INTRODUCTION

Infective endocarditis (IE) is regarded as a systemic septic disease in which vegetations containing bacterial masses are formed in the heart valve; affected patients exhibit various clinical symptoms due to heart failure (1). IE often develops in patients with underlying heart disease, and the patients may die of various complications if proper treatments are not performed (1). Oral streptococci are important causative agents of IE, and dental treatment has caused IE (2).

When transient bacteremia occurs in a patient with heart disease during or after dental treatment, oral streptococci adhere to and proliferate on abnormal heart valves to form vegetation (3). Dental treatments such as tooth extraction, periodontal surgery, implant surgery, and scaling are regarded as risk factors for IE (3). In addition, IE may be caused by bacteremia due to bleeding during tooth brushing (4). Bacteremia that occurs following dental treatments and tooth brushing is a result of bleeding from periodontal tissues; thus, the presence of periodontal disease increases the risk of such bleeding (5).

While periodontal disease is a known risk factor for IE, there is no evidence to support a direct relationship between the presence of dental caries and the onset of IE. Streptococcus mutans, a major pathogen of dental caries and a causative agent of IE, enters the bloodstream through bleeding from periodontal tissues (6). However, it remains unknown whether S. mutans from severe dental caries lesions can reach heart tissue through the bloodstream. S. mutans metabolizes sugar to produce acid and decalcifies enamel and dentin on the tooth surface (7). Subsequently, S. mutans can reach pulp, which is rich in nerves and capillaries (8). We previously reported that S. mutans is frequently isolated from inflamed pulp associated with severe dental caries (9). The most recent Japanese IE guidelines, to which we contributed, state that oral bacteria may enter the blood due to pulp exposure associated with severe caries (10).

To evaluate the role of pathogenic bacteria in the onset of IE, animal models have been used in which the heart valves of animals, such as rats or rabbits, are mechanically damaged using a catheter; bacteria are then administered into the blood by injection (11, 12). However, the role of pathogenic S. mutans in the onset of IE should be evaluated after S. mutans colonization of the oral cavity, because S. mutans is a commensal bacterial species in the oral cavity (13). As an animal model for reproducing human dental caries, a rat model of dental caries has been developed based on the provision of a caries-inducing diet and the administration of S. mutans into the oral cavity (14). In the present study, we developed a clinical model for evaluation of IE through concurrent induction of severe dental caries and heart valve injury. Using this novel animal model, we assessed whether S. mutans could travel from carious lesions to injured heart tissue via the exposed pulp.

RESULTS

Construction of a rat model with concurrent dental caries and heart valve injury.The schematic of the experimental protocol is shown in Fig. 1. S. mutans strain SA31R, a streptomycin-resistant substrain of SA31, was administered into the oral cavity of 45 rats aged 18 to 22 days to induce dental caries; 11 additional rats did not receive S. mutans. All 56 rats were fed a caries-inducing diet containing 56% sucrose until the end of the experimental period. Thirty-four of the 45 rats underwent heart valve injury at 90 days of age. Eleven rats were euthanized at 1 week after heart valve injury (age 97 days, injury A group); 11 rats were euthanized at 1 month after heart valve injury (age 120 days, injury B group); and the remaining 12 were euthanized at 3 months after heart valve injury (age 180 days, injury C group). The 11 rats who received S. mutans but did not undergo heart valve injury were prepared as the “no injury” group; these rats were euthanized at the same time as those in the injury C group. Finally, 11 rats who did not receive S. mutans but underwent heart valve injury were prepared as the “no infection” group; these rats were euthanized at the same time as those in the injury C group.

FIG 1
FIG 1

Schematic of the experimental protocol.

Analyses of oral condition.Representative images of dental caries of rats in each group are shown in Fig. 2A. Dental caries developed in the rats in a time-dependent manner, except in rats who did not receive S. mutans. When the groups with concurrent dental caries and heart valve injury (injury A, B, and C groups) were analyzed, plaque indices were significantly elevated in a time-dependent manner (P < 0.001) (Fig. 2B). In addition, the plaque index in the no infection group was significantly lower than the plaque indices in the injury B, injury C, and no injury groups (P < 0.05). Among the rats with concurrent severe dental caries and heart valve injury (injury A, B, and C groups), the numbers of S. mutans bacteria in mandibular bone were lowest in rats who were euthanized 1 week after heart valve injury (Fig. 2C); the number of S. mutans bacteria reached a plateau in the injury B group. No significant difference in the number of S. mutans bacteria in mandibular bone was observed between injury C and no injury groups, whereas no S. mutans bacteria were isolated from the no infection group.

FIG 2
FIG 2

Evaluation of oral condition. (A) Representative images of the teeth of rats in each group. Upper image: occlusal surface. Lower image: buccal surface. Bars, 1 mm. (B) Plaque indices. Data are expressed as the mean ± standard error (SE). Significant differences were determined by using analysis of variance (ANOVA) followed by Bonferroni posttest. *, P < 0.05 and ***, P < 0.001 between groups. (C) Average numbers of S. mutans bacteria in mandibular bone. Data are expressed as the mean ± SE. Significant differences were determined by using ANOVA followed by Bonferroni posttest. ***, P < 0.001 between groups. (D) Representative images of dental caries at each stage. Upper image: occlusal surface. Lower image: buccal surface. Bars, 500 μm. (E) Average numbers of teeth with dental caries extended to pulp. Data are expressed as the mean ± SE. Significant differences were determined by using ANOVA followed by Bonferroni posttest. *, P < 0.05 and ***, P < 0.001 between groups. (F) Average numbers of teeth with crown loss. Data are expressed as the mean ± SE. Significant differences were determined by using ANOVA followed by Bonferroni posttest. *, P < 0.05 and ***, P < 0.001 between groups.

Next, we classified the rats’ teeth into three different stages of dental caries severity: dental caries localized in enamel or dentin, dental caries extended to the pulp space, and severe dental caries resulting in loss of the tooth crown (Fig. 2D). Dental caries developed in a time-dependent manner, and the number of severe dental caries in the injury C group was highest among all groups (P < 0.05) (Fig. 2E and F). Although the injury C group and the no injury group differed only based on the presence or absence of heart valve injury, rats in the injury C group showed significant severe dental caries development compared with those in the no injury group (P < 0.05). When oral bacteria were compared between the injury C and no injury groups, Escherichia and Enterobacter were the main bacterial taxa identified in each group (Table 1). However, other bacterial taxa differed between the two groups; some bacterial taxa (e.g., Pasteurellaceae and Citrobacter) were observed only in rats with heart injury.

View this table:
TABLE 1

Specification of bacterial genera in oral specimens by sequence analysis using broad-range PCR

Analyses of heart condition.When the heart/body weight ratio was compared between rats for the same period, the heart/body weight ratio was significantly higher in the injury C group than that in the no infection group (P < 0.01) (Fig. 3A). Next, antigen-antibody assays were conducted using serum collected from rats and broth cultures of S. mutans bacteria to determine whether the bacteria had entered the bloodstream (Fig. 3B). Most serum samples from rats that had been infected with S. mutans for longer periods (i.e., the injury C and no injury groups) showed a positive reaction in the presence of S. mutans antigen. In contrast, the serum samples from rats that had been infected with S. mutans for shorter periods (i.e., the injury A and B groups) showed significantly lower positive reaction rates than serum samples from rats in the injury C and no injury groups (P < 0.05). No serum samples from the no infection group showed positive reactions. Among the injury A, B, and C groups, rates of S. mutans recovery from extirpated heart tissue increased in a time-dependent manner (Fig. 3C). Interestingly, S. mutans was isolated from heart tissue in nearly 20% of S. mutans-infected rats who had not undergone heart valve injury. In S. mutans-positive rats, the numbers of S. mutans in heart specimens ranged from 10 CFU to 1,000 CFU (Fig. 3D). The presence of Gram-positive bacteria was also confirmed by histopathological evaluation using Gram staining (Fig. 3E); however, no prominent abnormal findings were observed in histopathological evaluation using hematoxylin-eosin (HE) staining (Table 2). In addition, the bacteria present in heart specimens were positively stained using an anti-Cbm antibody for immunohistopathological analysis; this antibody had been previously raised against Cbm in the test bacteria (Fig. 3F). Positive anti-Cbm antibody staining results were not observed in heart specimens without the bacteria (Fig. 3G).

FIG 3
FIG 3

Evaluation of heart condition. (A) Heart to body weight ratio. Data are expressed as the mean ± SE. Significant differences were determined by using ANOVA followed by Bonferroni posttest. **, P < 0.01 between groups. (B) Antigen-antibody assay using serum samples collected from rats and broth cultures of S. mutans strain SA31R. Significant differences were determined by using ANOVA followed by Bonferroni posttest. *, P < 0.05, and **, P < 0.01 versus injury C group; #, P < 0.05, and ###, P < 0.001 versus no injury group. (C) Rates of S. mutans isolation from heart specimens. (D) Average numbers of S. mutans bacteria in heart specimens. Each closed circle represents the number of bacteria in a single rat’s heart specimen. Horizontal bars indicate mean values for the groups. (E) Representative image of Gram staining of transversely sectioned heart specimen. Right panel shows high-magnification image of the box on the left image. White arrowheads indicate bacterial masses. Representative image of immunohistochemical staining on transversely sectioned bacteria-positive (F) and bacteria-negative (G) heart specimens, using an anti-Cbm antibody. White arrowheads indicate bacterial masses.

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TABLE 2

Histopathological evaluation of extirpated heart tissue from rats

Analyses of other systemic conditions.We evaluated abnormal macroscopic findings in extirpated brain, lung, kidney, and spleen, all of which are affected by complications of IE (15–18). No abnormal findings were observed in the brains of any rats (see Fig. S1 in the supplemental material). Dot hemorrhage of the lung was observed in approximately half of the rats with concurrent severe dental caries and heart valve injury (injury A, B, and C groups) (see Table S1 and Fig. S2A in the supplemental material); this finding was less frequently found in rats with severe dental caries alone (no injury group) and in rats who had not received S. mutans (no infection group). Abnormal findings in spleen and kidney were observed in a small number of rats in the injury B and C groups (Table S1, Fig. S2B and C). The spleen/body weight ratio in rats euthanized at 1 month (age 120 days) after heart valve injury (injury B group) was significantly higher than the ratio in other groups (P < 0.001) (Fig. S2D).

Abnormal macroscopic findings of these tissues were mainly observed in rats with concurrent severe dental caries and heart valve injury; thus, histopathological evaluations were performed using HE staining for these tissues in the injury A, B, and C groups (see Table S2 in the supplemental material). Abnormal findings were mainly observed in bronchioles in the lung: infiltration of inflammatory cells, growth of respiratory epithelial cells, and death of respiratory epithelial cells showed high scores, which reached plateaus in the injury B group (see Table S2 and Fig. S3A and B in the supplemental material).

Correlations between dental caries and heart condition.We assessed the relationship between the stage of dental caries and the presence of S. mutans in heart specimens. Rats with S. mutans-positive heart specimens had significantly higher numbers of S. mutans bacteria in mandibular bone than did rats with S. mutans-negative heart specimens (P < 0.01) (Fig. 4A). In addition, rats with S. mutans-positive heart specimens had significantly more severe dental caries, such as caries that extended to pulp or resulted in loss of the tooth crown, relative to rats with S. mutans-negative heart specimens (P < 0.05) (Fig. 4B and C). Next, we assessed the relationship between the number of teeth with dental caries and the rate of S. mutans detection from heart tissue. Rats in whom ≥5 of 6 molars with dental caries extended to pulp had a significantly higher rate of S. mutans detection from heart tissue than that in rats who had ≤4 of 6 molars with this type of dental caries (Fig. 4D). Similarly, rats with ≥3 molars in which the crown was lost had a significantly higher rate of S. mutans detection from heart tissue than that in rats who had ≤2 molars with this type of dental caries (Fig. 4E).

FIG 4
FIG 4

Relationship between heart condition and dental caries status. Relationships between number of S. mutans bacteria in heart specimens and (A) number of S. mutans bacteria in mandibular bone, (B) average number of teeth with dental caries extended to pulp, and (C) average number of teeth with crown loss. Each closed circle represents the data for each rat. Horizontal bars indicate mean values for the groups. Severity of dental caries such as (D) dental caries extended to pulp and (E) crown loss due to dental caries and the respective effects on the detection rate of S. mutans bacteria from heart specimens. Number in parentheses below each bar graph indicates the number of rats. Significant differences were determined using the chi-square test. *, P < 0.05 and **, P < 0.01 between groups.

DISCUSSION

In vivo IE models have been constructed using small animals such as rats, in which the causative bacteria (including S. mutans) are generally administered via blood vessels (11, 12, 19, 20). However, for the most representative model, bacteria should be established at the location in the animals where those species are originally present, because most IE-causing bacteria are members of the indigenous flora for other parts of the human body (21, 22). Therefore, we performed an experiment in rats involving concurrent colonization of S. mutans in severe dental caries and injury of heart valves, in order to assess whether S. mutans in dental caries can reach the impaired heart tissue.

Most dental and oral surgical procedures (e.g., tooth extraction, dental scaling, periodontal surgery, and implant surgery) are known risk factors for the onset of bacteremia and IE (3), which occur as a result of bleeding from periodontal tissue. In addition, daily tooth brushing has been identified as a risk factor for the development of IE (4), especially in patients with severe periodontal disease (5). Unlike diabetes and myocardial infarction, which are closely related to sucrose intake, there have been no reports that sucrose intake is associated with the development of IE. However, S. mutans is regarded as a causative agent of IE; it colonizes the oral cavity mainly in the presence of sucrose. In addition, when sucrose intake causes severe dental caries with pulp exposure, oral bacteria could invade through exposed pulp capillaries and contribute to the development of IE in some instances. Therefore, we presumed that sucrose intake could be an indirect risk factor for IE through the initiation of severe dental caries; we planned the present study to test this hypothesis.

Dental pulp is located in the center of the tooth, surrounded by enamel and dentin, and consists of many nerves and blood vessels (8). S. mutans can be isolated from inflamed dental pulp tissue after decalcification of enamel and dentin (9). Therefore, we hypothesized that S. mutans within dental pulp tissue could travel to the heart tissue. In the present study, we combined two distinct rat models, a dental caries model (14) and a heart valve injury model (20). Using this experimental paradigm, we successfully evaluated whether S. mutans in dental pulp could reach heart tissue.

S. mutans is known to colonize the oral cavity during infancy (23). In rat dental caries models, S. mutans is administered into the oral cavity within a few weeks after birth to ensure S. mutans colonization (14). Dental caries can then be observed by provision of a caries-inducing diet, containing 56% sucrose, for 2 months (14). In the present study, we chose to continue feeding the caries-inducing diet for up to 6 months (depending on the point of euthanasia for each rat group) to induce severe dental caries; this period is longer than that used in previous studies. Therefore, the frequencies of teeth with dental caries that extended to pulp were approximately 20% after 3 months of the caries-inducing diet and 70% after 6 months of the caries-inducing diet. After 3 months of the caries-inducing diet, rats did not have severe dental caries involving loss of the tooth crown; conversely, after 6 months of the caries-inducing diet, rats exhibited loss of the tooth crown in approximately half of their teeth.

A previous study showed that the incidence of dental caries was significantly greater in patients with heart disease than in healthy participants (24). The high incidence of dental caries in the patients with heart disease might have been due to poor oral hygiene and high dietary intake of sucrose. In the present study, we examined three groups of rats, as follows: a group with concurrent severe dental caries and heart valve injury (injury C group), a group with only severe dental caries (no injury group), and a group with only heart valve injury (no infection group). These groups enabled comparison of dental caries status in the same dietary environment. The results of this study revealed that the presence of heart injury exacerbated dental caries, despite the absence of differences in oral hygiene and sucrose intake between rats with heart injury and healthy rats.

Because the number of S. mutans bacteria in the mandibular bone did not differ based on the presence or absence of heart injury, another mechanism might be responsible for the increased severity of dental caries due to heart injury. Therefore, we analyzed oral flora in these rats by sequencing analysis using broad-range PCR, which revealed that the presence or absence of heart valve injury did not affect the two most frequently detected oral bacterial taxa, Escherichia and Enterobacter. However, species in the Citrobacter and Pasteurellaceae taxa were frequently detected only in rats with injured heart valves. Species in the genus Citrobacter are regarded as keystone pathogens that can alter intestinal flora composition in mice (25). The appearance of Citrobacter spp. due to heart valve injury may induce changes in oral flora that increase susceptibility to dental caries. In addition, species in the family Pasteurellaceae exhibit urease activity and can survive in an acidic environment (26). Species in the family Pasteurellaceae were detected only in rats with injured heart valves, presumably because the oral cavities of these rats became acidic with tooth demineralization. The emergence of these types of bacteria may have caused important changes in the oral flora of rats with heart injury, which may have influenced the development of dental caries.

In the present study, rats were euthanized at 1 week, 1 month, or 3 months after injury to the aortic valve, and the presence or absence of S. mutans in the extirpated heart was evaluated. The rate of S. mutans isolation from heart specimens increased in a time-dependent manner after heart valve injury, which is consistent with the observation that IE induced by S. mutans mainly follows a subacute course (27). S. mutans may travel to heart tissue through capillaries in exposed pulp; this hypothesis was supported by S. mutans detection in an antigen-antibody assay using serum from rats with bacterial infection in heart tissue.

We used S. mutans in this study because this species is the only known causative bacterium for both dental caries and IE (20). In addition to S. mutans, Streptococcus sanguinis and Streptococcus mitis are known as major oral streptococci that cause subacute IE (10); however, these oral streptococci have low cariogenicity. Therefore, when assessing the effects of oral streptococci present in dental carious lesions on heart tissue in rats, these oral streptococci should be introduced via dental carious lesions caused by S. mutans.

For S. mutans isolation from rat heart tissue, we cut heart specimens into small pieces and homogenized them by sonication; the homogenates were serially diluted and plated on S. mutans-specific agar plates. Although we did not perform molecular biological analysis using these heart tissues, it is important to note that molecular biological analysis has become a useful tool for diagnosis of IE (10). In the present study, Gram staining and immunohistochemical staining were performed to identify bacteria present in rat heart tissue; in future studies, bacterial DNA amplification from heart tissues using molecular techniques may be an alternative effective method.

Interestingly, S. mutans was also detected in the heart tissues of rats with severe dental caries who had not undergone heart valve injury, although the frequency of this bacterial colonization was lower than that in rats with both severe dental caries and heart valve injury. Therefore, severe dental caries may be a risk factor for bacterial colonization in heart tissues, regardless of the presence of heart valve injury. Indeed, there have been some reports that IE may occur in patients without any heart disease (28). In support of this hypothesis, Staphylococcus aureus is a highly pathogenic bacterial species that can cause IE in patients without heart disease (29, 30). Among S. mutans strains, the SA31 strain used in the present study is highly pathogenic (21); thus, such strains may cause IE in the absence of heart valve injury.

As complications of IE, abnormal findings have been reported in the brain, lungs, kidneys, and spleen (15–18). In the present study, approximately 30% of the lungs had dot hemorrhages due to injury of the heart valve in the absence of S. mutans infection; this rate increased to approximately 50% in rats with concurrent S. mutans infection and heart valve injury. Based on these results, we presume that the presence of severe dental caries may aggravate disease in some other organs.

Increased infiltration of inflammatory cells, growth of respiratory epithelial cells, and death of respiratory epithelial cells in bronchioles are known parameters for assessment of pneumonia (31, 32). IE can be associated with pneumonia; thus, we focused on these histopathological assessments in the lung (10). We found that the levels of these histopathological parameters reached a plateau at 1 month after heart injury and were reduced at 3 months after heart injury. Therefore, we concluded that severe dental caries and heart injury may cause transient pneumonia. In addition, spleen enlargement is common in subacute IE, which constitutes approximately 20% of all reported cases of IE (10). In the present study, the spleen weight was highest at 1 month after heart valve injury. Our results suggest that disease progression differs between the heart and other organs. Although the causative mechanisms remain unknown, the results of the present study may provide meaningful data for treatment of IE in clinical practice.

Although a large number of studies have focused on the relationship between IE and dental procedures (33), to the best of our knowledge, there have been no studies regarding the relationship between S. mutans colonization in heart tissue and in severe dental caries. The rates of S. mutans colonization of heart tissues were not high in any group, although the detection frequencies were extremely high relative to the commonly known IE frequency of 1 to 5 in 100,000 people (34). In the present study, rats with heart injury had severe dental caries, such that most of the teeth exhibited dental caries extending to pulp or resulting in loss of the tooth crown; approximately 50% of these rats had S. mutans in heart tissue. In rats with comparatively milder dental caries, S. mutans was isolated from heart specimens at a much lower frequency (<10%), despite the presence of heart injury. Based on our results, we suspect that the severity of dental caries may be related to the detection rate of S. mutans in heart tissue.

In summary, our findings revealed that S. mutans present in severe dental caries lesions could reach heart tissue; this mainly occurred in rats with heart valve injury, but it sometimes occurred in rats without heart valve injury. In addition, heart injury induced changes in oral flora, which may have been associated with the development of dental caries. Furthermore, rats with concurrent severe dental caries and heart valve injury also exhibited transient abnormalities in other organs, such as spleen enlargement and lung inflammation.

MATERIALS AND METHODS

Ethics statement.All rats were treated humanely in accordance with the guidelines of the National Institutes of Health and the AERI-BBRI Animal Care and Use Committee. All animal experiments were approved by the Institutional Animal Care and Use Committee of Osaka University Graduate School of Dentistry (approval no. 24-019-0).

S. mutans strains and growth conditions.S. mutans strain SA31 was used (35); it was confirmed to be S. mutans based on observation of rough colony morphology on Mitis Salivarius agar (Difco Laboratories, Detroit, MI) plates containing bacitracin (0.2 U/ml; Sigma Chemical Co., St. Louis, MO) and 15% (wt/vol) sucrose (MSB agar), as well as 16S rRNA sequence analysis with the primers 8UA (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1540R (5′-AAG GAG GTG ATC CAG CC-3′), as described previously (36). Streptomycin resistance was elicited in SA31 by repeated passage in increasing concentrations of the antibiotic prior to the animal experiment (as in a prior study [37]), up to a final concentration of 1,500 μg/ml streptomycin; this resistant strain was termed SA31R. For routine growth, SA31R was grown in brain heart infusion broth (Difco Laboratories), as well as on MSB agar containing 1,500 μg/ml streptomycin, as used for selection.

Schematic of the experimental protocol.The schematic of the experimental protocol is shown in Fig. 1. Dental caries was induced using a previously described method (14). Briefly, 56 Sprague-Dawley male rats aged 15 to 18 days were fed normal diet CE-2 (CLEA Japan, Osaka, Japan) containing tetracycline (4 mg/g) and given water containing penicillin G (4,000 U/ml) before the establishment of the inoculated organism in the oral cavity. These rats were then fed a caries-inducing diet containing 56% sucrose (CLEA Japan) until the end of the experimental period, in order to induce severe dental caries. At 18 days of age, S. mutans strain SA31R (1 × 108 CFU) was administered into the oral cavity of 45 rats (in each rat, once per day for 5 days); the remaining 11 rats did not receive S. mutans. One week after infection, dental plaque was collected from each rat using a sterilized cotton swab; plaque samples were seeded in MSB agar containing 1,500 μg/ml streptomycin, and all rats infected with S. mutans were confirmed to exhibit S. mutans colonization in the oral cavity. Subsequently, the heart valves of 34 of the 45 S. mutans-infected rats, as well as the heart valves of all noninfected rats, were injured when the rats reached 90 days of age, using a previously described method (21). Briefly, these rats were anesthetized with a mixture of xylazine and midazolam (0.1 ml/100 g). A sterile polyethylene catheter with a guide wire was surgically placed across the aortic valve of each rat via the right carotid artery, and the tip was positioned and placed at the aortic valve in the left ventricle. Among the 34 rats with concurrent S. mutans infection and injured heart valves, 11 were euthanized at 1 week after heart valve injury (age 97 days), 11 were euthanized at 1 month after heart valve injury (age 120 days), and the remaining 12 were euthanized at 3 months after heart valve injury (age 180 days). Eleven rats without S. mutans infection and 11 rats without heart valve injury were euthanized at the age of 180 days.

Recovery of S. mutans from mandibular bones.Recovery of S. mutans strain SA31R from mandibular bone was evaluated in accordance with a previously described method (38). After the rats were euthanized, their mandibular bones were removed and divided into two pieces of equal size. Left mandibular bones were placed in sterile saline, and bacteria were separated from these bones by sonication. The resulting bacterial suspension was serially diluted with sterile saline and cultured on MSB agar plates containing 1,500 μg/ml streptomycin. After incubation at 37°C for 48 h, the numbers of colonies on the agar plates were counted to determine the numbers of S. mutans bacteria present in the mandibular bones.

Evaluation of oral condition.Plaque indices were calculated using the method of Quigley and Hein (39). Briefly, plaque on the buccal, occlusal, and lingual surfaces of right and left maxillary molar teeth of rats (six teeth per rat)—which were extirpated after rats had been euthanized—was stained with Red-Cote (John O. Butler Co., Chicago, IL) and each surface was classified into three grades (grades 0 to 3). The plaque index was obtained by dividing the total grade by the number of surfaces of teeth.

The severity of dental caries was evaluated by observation of the occlusal surfaces of right maxillary and mandibular molar teeth (six teeth per rat) by a dentist using a stereomicroscope. The presence or absence of pulp exposure was determined by the dentist using a sterilized 27-gauge needle (0.4 mm diameter) (Terumo Co., Tokyo, Japan) under a stereomicroscope. The severity of dental caries was classified into the following three levels: dental caries localized in enamel or dentin, dental caries extended to pulp space, and severe dental caries resulting in loss of the tooth crown. Among teeth with pulp exposure, crown loss was defined as the loss of all cusps and a crown height lower than that of the original crown when teeth were observed from buccal and lingual sides using a stereomicroscope.

Identification of oral bacteria by broad-range PCR.To define the major bacterial taxa present in dental plaque specimens, sequence analysis was conducted with broad-range PCR assays targeting eubacterial 16S rRNA with the primers PA (5′-AGA GTT TGA TCC TGG CTC AG-3′) and PD (5′-GTA TTA CCG CGG CTG CTG-3′); bacterial DNA samples used in this assay were extracted from dental plaque at the time of euthanasia, as previously described (40). Briefly, amplified DNA fragments were extracted from agarose gels using a Qiaex II gel extraction kit (Qiagen, Hilden, Germany) and directly cloned into the pGEM-T Easy vector (Promega, Madison, WI). Sequence analyses were performed using the BigDye Terminator v3.1 (Thermo Fisher Scientific, Waltham, MA), BigDye Xterminator (Thermo Fisher Scientific), and 3130xl genetic analyzer (Thermo Fisher Scientific); all analyses were performed by Fasmac Co., Ltd. (Kanagawa, Japan). The resulting 16S rRNA sequences were compared with sequences available in the GenBank, EMBL, and DDBJ databases using the gapped BLAST 2.0.5 program obtained from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/BLAST/).

Antigen-antibody assay.To confirm that S. mutans strain SA31R had entered the blood of rats, an antigen-antibody assay was performed with serum collected from the rats and with S. mutans strain SA31, using a previously described method with some modifications (41). The density of a broth culture of S. mutans strain SA31R was adjusted to an optical density at 550 nm (OD550) of 1.0 with phosphate-buffered saline; an aliquot of the broth culture and an aliquot of serum (10 μl each) were mixed on a microscope slide and incubated for 10 min at room temperature. Then, a positive reaction to S. mutans strain SA31R, indicative of anti-S. mutans antibodies in blood, was regarded as the development of agglutination in the mixture on the microscope slide. As a negative control, phosphate-buffered saline and serum were mixed on a microscope slide.

Recovery of S. mutans from heart specimens.Extirpated rat hearts were sectioned transversely relative to the aortic valves. Half of the heart tissue from each rat was aseptically cut into small pieces, which were placed in phosphate-buffered saline and homogenized by sonication. Then, suspensions containing the homogenized tissue were serially diluted and placed onto MSB agar plates containing 1,500 μg/ml streptomycin. After incubation at 37°C for 48 h, the numbers of colonies on agar plates were counted to determine the numbers of bacteria present in the heart specimens.

Evaluation of extirpated tissues.Macroscopic abnormal findings of extirpated heart, brain, lung, kidney, and spleen tissues were evaluated by a senior laboratory animal technician for the entire study. The body weight before euthanasia and the weight of the extirpated spleen were measured, and the ratio of spleen weight to body weight was determined as an indicator of systemic inflammation (42).

Half of the extirpated heart tissue from each rat, as well as all other extirpated tissues from each rat, were fixed in 10% formalin neutral buffer solution (Fujifilm Wako Pure Chemical Corporation, Tokyo, Japan), embedded in paraffin, and cut into 3-μm sections. HE staining was then performed using these sections, followed by evaluation of pathological features, as shown in Table 2 and Table S2. Histopathological observations were evaluated by scoring as follows: 0 (none), 1 (mild), 2 (moderate), and 3 (severe), in accordance with the method used in a previous study (21). All scoring evaluations were performed in a double-blinded fashion by a pathologist (Sept Sapie Co., Ltd., Tokyo, Japan).

Immunohistochemical staining of sections of heart tissue was performed using the following procedure, which was described in a previous publication (43). For antigen retrieval, heart tissue sections were incubated with RM102-C (LSI Medience, Tokyo, Japan) at 105°C for 1 h. Then, the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used in accordance with the manufacturer’s instructions. First, heart tissue sections were blocked with 3% H2O2 and 2.5% horse serum; then, sections were incubated with anti-Cbm antibody (21), diluted 100-fold with phosphate-buffered saline, at 4°C for 12 h. Subsequently, sections were incubated with secondary antibody from the kit at room temperature for 30 min. In addition, counterstaining was performed with hematoxylin solution.

Statistical analysis.Statistical analyses were performed using Prism 6 (GraphPad Software Inc., La Jolla, CA). Comparisons between two groups were performed using the chi-square test. Intergroup differences in each analysis were determined by using analysis of variance (ANOVA). Bonferroni correction was used for post hoc analysis. Regression analysis was performed to compare correlations between two groups. A P value of <0.05 was considered to be statistically significant.

ACKNOWLEDGMENTS

We thank Rewa Yanagisawa, Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, for technical support with molecular analyses. Histopathological evaluations were advised by Naoki Iwashita, Department of Pharmacology, School of Veterinary Medicine, Azabu University. We thank Ryan Chastain-Gross from Edanz Group for editing a draft of the manuscript.

This work was supported by JSPS KAKENHI grants JP18H03010, JP18K09831, and JP18K17254.

FOOTNOTES

    • Received 3 December 2019.
    • Returned for modification 3 January 2020.
    • Accepted 11 April 2020.
    • Accepted manuscript posted online 20 April 2020.

  • Supplemental material is available online only.

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KEYWORDS

Streptococcus mutans
dental caries
Dental Pulp
heart valve
infective endocarditis
rat model

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