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The involvement of oral bacteria in the pathogenesis of cardiovascular diseases has been the focus of attention in many studies, and several periodontal pathogens have been detected in diseased cardiovascular lesions, suggesting relationships between oral microorganisms and cardiovascular diseases. However, no information is available regarding the involvement of cariogenic pathogens such as Streptococcus mutans. The presence of oral streptococcal species and periodontitis-related bacteria in 35 heart valve and 27 atheromatous plaque clinical specimens, as well as 32 dental plaque specimens from the same subjects, was analyzed using a PCR method. Furthermore, broad-range PCR with DNA sequencing analysis was employed to identify the bacterial species in those samples. Streptococcus mutans was frequently detected in the heart valve (69%) and atheromatous plaque (74%) specimens, while other bacterial species, including those related to periodontitis, were detected with much lower frequencies. The bacterial composition in cardiovascular tissues was found to be markedly distinct from that in dental plaque, with only a limited number of species, including S. mutans, in the cardiovascular regions shown to have possibly originated from the oral cavity. Semiquantitative assay results revealed that S. mutans was detected in significant quantities in the heart valve (40%) and atheromatous plaque (48%) specimens, whereas the quantities of all other tested bacterial species, including several related to periodontitis, were negligible in the cardiovascular samples. These results indicate that S. mutans is a possible causative agent of cardiovascular disease.
Accumulated evidence suggests that oral bacterial pathogens are associated with several kinds of systemic diseases, such as infective endocarditis (IE), cardiovascular diseases, bacterial pneumonia, low birth weight, and diabetes mellitus (12). Those associations are speculated to be initiated by transient or prolonged bacteremia caused by oral infection: i.e., from professional dental treatments and daily oral care practices such as tooth brushing and flossing, as well as from food chewing, which possibly induces dissemination of oral bacteria into the bloodstream (21). Oral streptococcal species are major components of the oral microflora that are known to occasionally cause bacteremia and IE (13). Streptococcus mutans, a major pathogenic agent of dental caries, has also been isolated from the blood of patients with IE, strongly suggesting a close relationship of the pathogen with IE (5, 23, 24).
The recent development of several molecular techniques has enabled prompt identification of targeted bacterial species in specimens with significantly improved specificity and sensitivity. PCR methods using primers constructed with a species-specific nucleotide alignment are widely used for the detection of specific species. In addition, broad-range eubacterial PCR with amplification of bacterial DNA and subsequent direct sequencing is considered to be a reliable diagnostic tool (18, 19).
Dental caries and chronic marginal periodontitis are two major infectious diseases clinically encountered in the field of dentistry. Recently, several studies have reported detection of periodontal pathogens in cardiovascular specimens from patients using a PCR method, suggesting relationships between oral microorganisms and systemic cardiovascular diseases (20). However, there are no reports of oral streptococci detected in those tissues, especially regarding cariogenic S. mutans, which is a potential cause of IE. In the present study, we analyzed the presence of streptococcal species in diseased heart valve and atheromatous plaque specimens, as well as in dental plaque samples from the same subjects.
A total of 35 heart valve specimens from 24 males and 11 females (average age, 67.4 years old; range, 46 to 84 years) and 27 atheromatous plaque specimens from 22 males and 5 females (average age, 70.6 years old; range, 45 to 81 years) were collected according to a protocol approved by the Ethics Committee of Osaka Rosai Hospital. All of the heart valve tissue specimens were excised during a valve replacement procedure, following diagnosis of aortic regurgitation, mitral regurgitation, or tricuspid regurgitation, while the atheromatous plaque specimens were collected during treatment for a thoracic or abdominal aortic aneurysm. These specimens were aseptically obtained at the Department of Cardiovascular Surgery, Osaka Rosai Hospital, Sakai, Osaka, Japan, from December 2004 to November 2005. In addition, supra- and subgingival plaque samples from 32 of those patients were taken from the mesial and buccal subgingival sites of all teeth with sterile Gracey curettes at the Department of Dentistry and Oral Surgery of Osaka Rosai Hospital prior to the cardiovascular operations.
Whole DNA fractions were extracted from the heart valve and atheromatous plaque specimens, after being aseptically cut into small pieces, as well as from the dental plaque samples using a method described previously (16). Thereafter, a PCR method using specific primer sets for the glucosyltransferase gene was employed to detect six oral streptococcal species (S. mutans, Streptococcus sobrinus, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus oralis, and Streptococcus gordonii) as described previously (9). Briefly, PCR was carried out in 20 μl of a reaction mixture containing 0.5 U of TaKaRa Ex Taq (TAKARA BIO, Inc., Otsu, Shiga, Japan), 0.5 μM of oligonucleotide primers, template DNA, and 1.5 mM of MgCl2, according to the manufacturer's protocols. Amplification was performed with a GeneAmp 9700 thermal cycler (Applied Biosystems, Foster City, Calif.) using the following parameters: 30 cycles of a denaturing step at 98°C for 10 s and primer-annealing and extension steps at 66°C or 70°C for 1 min. The primer-annealing and extension temperatures were changed for each primer set (Table (Table1).1). S. mutans MT8148, S. sobrinus 6715, S. salivarius HHT, S. sanguinis ATCC 10556, S. oralis ATCC 10557, and S. gordonii ATCC 10558 were used as positive controls. In addition, the periodontitis-related bacterial species Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, Tannerella forsythia, Actinobacillus actinomycetemcomitans, and Campylobacter rectus were analyzed using a PCR method, as described previously (1). This PCR assay was done by the method described above, except for the thermal cycles, which were as follows. Initial denaturation was performed at 95°C for 30 s, followed by 55°C or 60°C for 30 s and 72°C for 1 min, with a final extension step at 72°C for 7 min. The annealing temperature was varied depending on the primer set used (Table (Table11).
A broad-range PCR technique targeting 16S rRNA with direct sequencing was carried out to quantitatively identify bacterial species in the specimens. 16S rRNA was amplified by PCR using AmpliTaq Gold polymerase (Applied Biosystems) with the broad-range 16S rRNA primers 536f and 1050r (19). Next, the PCR products were separated by electrophoresis on 1.5% agar gel and the amplified DNA fragments were extracted using a QIAEX II gel extraction kit (QIAGEN Sciences, Düsseldorf, Germany). The extracted DNA was directly cloned into a pGEM-T Easy vector (Promega, Madison, Wis.), and then 10 clones from each sample were randomly chosen and analyzed by dye terminator reaction with a DNA sequencing system (ABI PRISM 310 Genetic Analyzer; Applied Biosystems) and the BigDye terminator cycle sequencing kit. Data analysis was performed using Gene Works software (IntelliGenetics, Mountain View, Calif.). To identify the bacterial species, the 16S rRNA sequences were compared with those available in the GenBank, EMBL, and DDBJ databases, using the gapped BLASTN 2.0.5 program obtained from the National Center for Biotechnology Information server (http:/www.ncbi.nlm.nih.gov/BLAST/). Identification at the species level was determined when the 16S rRNA sequence similarity was greater than 99% of that of the GenBank prototype strain sequence, while identification at the genus level was defined as a 16S rRNA sequence similarity of 97% with that of the GenBank prototype strain sequence. The number of species identified in the 10 random clones was converted to a semiquantitative amount of species per sample.
Fisher's exact probability test was used for statistical analyses of the comparative frequencies of bacterial occurrence. A P value of <0.05 was considered significant.
The bacterial compositions in the 62 cardiovascular specimens (35 heart valve and 27 atheromatous plaque specimens) were compared to those in dental plaque samples from the same subjects (Table (Table2).2). S. mutans was most prevalent in the heart valve tissue (68.6%) and atheromatous plaque (74.1%) specimens and was also significantly distributed in the dental plaque samples (88.9 to 100%). In contrast, S. sanguinis had the second greatest occurrence in dental plaque (77.8 to 85.7%), while it was detected at a low frequency in the heart valve (17.1%) and atheromatous plaque (25.9%) specimens. The other species, including S. sobrinus, S. salivarius, and S. oralis, were rarely detected in the heart valve and atheromatous plaque specimens, though S. oralis and S. salivarius were prevalent in dental plaque at ratios ranging from 71.4% to 72.2% and from 16.7% to 21.4%, respectively. As for periodontitis-related bacteria, T. denticola was the most frequently detected species in heart valve tissues (40.0%) and atheromatous plaque (44.4%). A. actinomycetemcomitans, the second most frequently detected species, was detected in 25.7% of the heart valves and 25.9% of atheromatous plaque samples. In addition, the detection frequencies of P. gingivalis, P. intermedia, and C. rectus were extremely low in both the heart valve and atheromatous plaque specimens, while T. forsythia was not detected in any of those, in spite of its high detection frequency in dental plaque (85.7 to 94.4%). In contrast, all six of the periodontitis-related bacteria tested were detected in dental plaque (16.7 to 100%). These results indicate that the bacterial compositions in cardiovascular tissues were distinct from those in dental plaque, with only selected species found in the cardiovascular region, including S. mutans, T. denticola, and A. actinomycetemcomitans.
We also semiquantitatively analyzed the various bacterial components in the cardiovascular specimens using a broad-range PCR method with sequencing analysis. As for the heart valve tissues, streptococcal species were detected in 77.8% of the subjects and were found to account for 59.4% of the bacterial population (Table (Table3).3). Among them, S. mutans was found to be significantly prevalent, with a detection frequency of 77.8% in the subjects and was prevalent in 40.0% of them. Other streptococcal species, including S. sanguinis, S. oralis, S. gordonii, S. pneumoniae, S. mitis, S. thermophilus, S. vestibularis, S. salivarius, and S. cristatus, were also detected in 38.9% of the subjects and were prevalent in 19.4% of the populations, though each of those species had a significantly lower detection frequency than S. mutans. Other species were also identified, though their incidence rates were far below that of S. mutans. Dental plaque samples from the same subjects were also quantitatively analyzed, and various species also found in the heart valve tissues were detected. Although streptococcal species were frequently detected, their quantities were quite different from those in the heart valve specimens. In addition, periodontitis-related bacteria were detected in significant quantities and frequency in dental plaque, whereas their presence in heart valve tissue was shown to be negligible by this method.
We also semiquantitatively analyzed the atheromatous plaque specimens (Table (Table4).4). Similar to those from the heart valve, streptococcal species were most frequently detected (frequency, 88.9%; quantity, 81.1%), with S. mutans the most predominant species (77.8% and 48.1%, respectively). The incidence of other species, including periodontitis-related bacteria, was negligible compared to that of S. mutans. From the results of our analysis of dental plaque samples, the bacterial profiles for frequency and quantity were found to be significantly different from those of atheromatous plaque.
S. mutans is known to be one of the pathogens that causes IE, though it is primarily a major cariogenic pathogen that is a normal inhabitant of the oral cavity in most individuals. Unexpectedly, S. mutans was detected at high frequencies and quantities in both heart valve tissues and atheromatous plaque samples in the present study, as a simple PCR analysis detected it significantly more frequently than T. denticola (Tables (Tables2),2), which is a periodontal bacterium most frequently detected in atherosclerotic lesions (17). In addition, our semiquantitative analysis revealed that S. mutans was significantly prevalent in both quantity and frequency in heart valve and atheromatous plaque specimens, as compared to the other tested species, and while the amounts of periodontitis-related bacteria including T. denticola were negligible (Tables (Tables33 and and4).4). Although there are limits to interpreting our semiquantitative analysis results from 10 clones for each sample in this study, the broad-range PCR and sequencing method used is able to identify all of the species registered in available databases, which supports our finding that S. mutans was more frequently detected than any other species, in addition to the results of the simple PCR method. Therefore, it is possible to speculate that S. mutans is a causative agent for cardiovascular disease.
Periodontitis-related bacteria have been identified in atheromatous samples using PCR in several studies, in which the detection rates varied, likely due to differences in the DNA extraction methods employed. Haraszthy et al. (7) reported the detection frequencies of periodontitis-related bacteria in 50 atheromatous specimens, which included cytomegalovirus (38%), Chlamydia pneumoniae (18%), T. forsythia (30%), P. gingivalis (26%), A. actinomycetemcomitans (18%), and P. intermedia (14%). Another study that analyzed carotid endarterectomy samples by immunostaining showed that C. pneumoniae, cytomegalovirus, herpes simplex virus 1, P. gingivalis, and S. sanguinis were positive, with frequencies ranging from 9% to 64% (4). In addition, T. denticola was detected in 23% of DNA samples of formalin-fixed, paraffin-embedded atherosclerotic lesions, such as thoracic and abdominal aneurysms (17). However, oral streptococcal species were not targeted for detection in those studies, and to the best of our knowledge, this is the first study to analyze streptococcal profiles in both dental plaque and cardiovascular tissue specimens from the same subjects. T. denticola was the most frequently detected species among six major periodontitis-related bacteria (Table (Table2),2), though our broad-range PCR assays revealed that S. mutans was predominant compared to the other tested periodontitis-related bacteria in aneurysm tissues.
Systemic challenge by P. gingivalis was reported to accelerate atherogenic plaque formation in a murine model (11). One of the crucial steps for the development of atheromatous plaque lesions is formation of foam cells, which are macrophages that accumulate from excess cholesterol, and P. gingivalis has been shown to enhance their formation (10). Interestingly, that study also showed that S. mutans strain GS-5 possessed properties similar to those of P. gingivalis. In addition, P. gingivalis and S. mutans were shown to induce platelet aggregation, which presumably leads to thrombus formation (8), while it was also recently found that S. mutans cells bind to extracellular matrix molecules and fibrinogen with contribution from the major surface protein antigen PAc (3). It is unknown if S. mutans can invade endothelial cells and form atheromatous lesions, though those previous findings suggest an etiological involvement of the bacterium in cardiovascular diseases. Additional studies are necessary to provide the biological rationale for the involvement of this cariogenic pathogen.
The broad-range PCR and direct sequencing as well as conventional PCR techniques used in this study are reliable methods also utilized for clinical diagnosis of IE (18, 19). From the present findings obtained with those methods, an important question is the source of S. mutans found in cardiovascular tissues. This bacterium is a normal oral inhabitant that may migrate to cardiovascular endothelial tissues. In previous reports, several blood isolates of S. mutans from IE patients were shown to possess cariogenic properties and were considered to be oral derivatives (14, 15). In future studies, it will also be necessary to determine if the S. mutans organisms found in cardiovascular tissues are cariogenic strains. In addition, though diagnoses for cardiovascular diseases are well characterized, there is no specific classification of disorders related to bacterial occurrence. Thus, extensive investigations are needed to answer these critical questions.
This study was supported by a part of the 21st Century COE program entitled “Origination of Frontier BioDentistry” at Osaka University Graduate School of Dentistry, supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Grant-in-Aid for Scientific Research (B) 16390605 from the Japan Society for Promotion of Science.