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Molecular analysis of oral bacteria in dental biofilm and atherosclerotic plaques of patients with vascular disease

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    Clarissa Pessoa Fernandes
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    Corresponding author at: Rua Tomás Acioli, 1100, ap. 603, Joaquim Távora, Fortaleza, Ceará CEP: 60135180, Brazil. Tel.: +55 85 96759825, +55 85 32462950.
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    1 This author takes responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation.
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    Department of Stomatology and Oral Pathology, School of Dentistry, Federal University of Ceará, Fortaleza, Ceará, Brazil
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    Francisco Artur Forte Oliveira
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    Department of Stomatology and Oral Pathology, School of Dentistry, Federal University of Ceará, Fortaleza, Ceará, Brazil
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    Paulo Goberlânio de Barros Silva
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    Department of Stomatology and Oral Pathology, School of Dentistry, Federal University of Ceará, Fortaleza, Ceará, Brazil
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    Ana Paula Negreiros Nunes Alves
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    Department of Oral Pathology, School of Dentistry, Federal University of Ceará, Fortaleza, Ceará, Brazil
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    Mário Rogério Lima Mota
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    Department of Stomatology and Oral Pathology, School of Dentistry, Federal University of Ceará, Fortaleza, Ceará, Brazil
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    Raquel Carvalho Montenegro
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    Human Cytogenetics Laboratory, Institute of Biological Sciences, Federal University of Para, Belém, Pará, Brazil
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    Rommel Mario Rodriguez Burbano
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    Human Cytogenetics Laboratory, Institute of Biological Sciences, Federal University of Para, Belém, Pará, Brazil
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    Aline Damasceno Seabra
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    Institute of Biological Sciences, Federal University of Para, Belém, Pará, Brazil
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    José Glauco Lobo Filho
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    School of Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazil
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    Danilo Lopes Ferreira Lima
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    School of Dentistry, University of Fortaleza, Fortaleza, Ceará, Brazil
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    Antônio Wilon Evelin Soares Filho
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    Hospital de Messejana Dr. Carlos Alberto Studart Gomes, Fortaleza, Ceará, Brazil
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    Fabrício Bitu Sousa
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    Department of Stomatology and Oral Pathology, School of Dentistry, Federal University of Ceará, Fortaleza, Ceará, Brazil
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Open AccessPublished:April 26, 2014DOI:https://doi.org/10.1016/j.ijcard.2014.04.201

      Abstract

      Background

      Oral bacteria have been detected in atherosclerotic plaques at a variable frequency; however, the connection between oral health and vascular and oral bacterial profiles of patients with vascular disease is not clearly established. The aim of this study was to evaluate the presence of oral bacterial DNA in the mouth and atherosclerotic plaques, in addition to assessing the patients’ caries and periodontal disease history.

      Methods

      Thirty samples of supragingival and subgingival plaque, saliva and atherosclerotic plaques of 13 patients with carotid stenosis or aortic aneurysm were evaluated, through real-time polymerase chain reaction, for the presence of Streptococcus mutans (SM), Prevotella intermedia (PI), Porphyromonas gingivalis (PG) and Treponema denticola (TD). All patients were submitted to oral examination using the DMFT (decayed, missing and filled teeth) and PSR (Periodontal Screening and Recording) indexes. Histopathological analysis of the atherosclerotic plaques was performed.

      Results

      Most of the patients were edentulous (76.9%). SM, PI, PG and TD were detected in 100.0%, 92.0%, 15.3% and 30.7% of the oral samples, respectively. SM was the most prevalent targeted bacteria in atherosclerotic plaques, detected in 100% of the samples, followed by PI (7.1%). The vascular samples were negative for PG and TD. There was a statistically significant difference (p < 0.05) between the presence of PG and TD in the oral cavity and vascular samples.

      Conclusion

      SM was found at a high frequency in oral and vascular samples, even in edentulous patients, and its presence in atherosclerotic plaques suggests the possible involvement of this bacterium in the disease progression.

      Keywords

      To the Editor:
      The connection between periodontal and vascular disease has been investigated, and periodontal microorganisms have been identified in atherosclerotic plaque samples at a variable frequency, which suggests that these bacteria can be related to the etiopathogenesis of atherosclerosis. Few studies have investigated the presence of cariogenic microorganisms, such as Streptococcus mutans (S. mutans), in vascular tissue, although these bacteria are capable of invading endothelial cells and stimulating the production of inflammatory markers, in addition to being detected at a high frequency in these lesions [
      • Nakano K.
      • Nemoto H.
      • Nomura R.
      • et al.
      Detection of oral bacteria in cardiovascular specimens.
      ,
      • Kozarov E.
      • Sweier D.
      • Shelburne C.
      • Progulske-Fox A.
      • Lopatin D.
      Detection of bacterial DNA in atheromatous plaques by quantitative PCR.
      ,
      • Nakano K.
      • Inaba H.
      • Nomura R.
      • et al.
      Detection of cariogenic Streptococcus mutans in extirpated heart valve and atheromatous plaque specimens.
      ].
      Furthermore, detailed evaluation of oral health status of all patients enrolled in the study, that has been performed only by a few authors and in a small group of patients [
      • Cairo F.
      • Gaeta C.
      • Dorigo W.
      • et al.
      Periodontal pathogens in atheromatous plaques. A controlled clinical and laboratory trial.
      ], could provide evidence of a connection between oral diseases and the frequency of oral microorganisms in vascular samples. Thus, to evaluate the presence of DNA of cariogenic and periodontopathogenic bacteria in the mouth and in atherosclerotic plaque samples of the same patients, in addition to evaluating their oral health status, 13 patients with carotid stenosis or aortic aneurism were enrolled in this study.
      All patients underwent bedside oral examination, which was performed by two previously calibrated examiners (Kappa values: 0.80 to 0.97), up to one day before surgery. The indexes DMFT and PSR were used to evaluate caries history and periodontal condition respectively. All participants gave their informed consent, and the study was approved by the Ethics Committee of Hospital Universitário Walter Cantídio of the Federal University of Ceara.
      Supragingival and subgingival plaque samples were collected for dentate patients, according to a previously reported protocol [
      • Cairo F.
      • Gaeta C.
      • Dorigo W.
      • et al.
      Periodontal pathogens in atheromatous plaques. A controlled clinical and laboratory trial.
      ]. For edentulous patients, saliva samples were collected (Supplemental methods).
      A total of 14 atherosclerotic plaques were collected, aseptically, during endarterectomy surgical procedures from the carotid artery or surgery of abdominal or thoracic aortic aneurysm. All oral and vascular samples were stored in a sterile vial containing phosphate-buffered saline (PBS) at −20 °C for further analysis using real-time PCR. Fragments of vascular samples were fixed in 10% formalin for histopathological analysis.
      Total DNA was extracted from each sample with a protocol based on the cetyltrimethylammonium bromide method [
      • Wilson K.
      Preparation of genomic DNA from bacteria.
      ]. A total of 30 samples of DNA extracted from saliva, supragingival plaque, subgingival plaque and atherosclerotic plaque were subjected to real-time PCR (Supplemental methods) for the detection of DNA from 4 different bacterial species: S. mutans, Porphyromonas gingivalis, Prevotella intermedia and Treponema denticola. TaqMan probes to 16S bacterial ribosomal DNA (Supplemental Table 1) were specifically designed for this study (Life Technologies).
      Data regarding demographic characteristics, vascular disease, oral health status and medical history of the patients are summarized in Table 1. The mean number of missing teeth was high, and most of the patients included in the present study were edentulous. Edentulous patients were included in the present study because in previous studies, even when these patients were considered to be part of a control group, samples of atherosclerotic plaques were positive for oral bacteria [
      • Kozarov E.
      • Sweier D.
      • Shelburne C.
      • Progulske-Fox A.
      • Lopatin D.
      Detection of bacterial DNA in atheromatous plaques by quantitative PCR.
      ,
      • Zaremba M.
      • Górska R.
      • Suwalski P.
      • Czerniuk M.R.
      • Kowalski J.
      Periodontitis as a risk factor of coronary heart diseases?.
      ]. Furthermore, of the studies that have included edentulous patients, none examined the oral bacterial profile of these patients [
      • Kozarov E.
      • Sweier D.
      • Shelburne C.
      • Progulske-Fox A.
      • Lopatin D.
      Detection of bacterial DNA in atheromatous plaques by quantitative PCR.
      ,
      • Zaremba M.
      • Górska R.
      • Suwalski P.
      • Czerniuk M.R.
      • Kowalski J.
      Periodontitis as a risk factor of coronary heart diseases?.
      ].
      Table 1Demographic, clinic and dental characteristics of patients with carotid stenosis and aortic aneurysm. Fortaleza — CE, Brazil, 2013.
      VariablesSamples
      Demographic13 patients
       Age68.5 ± 10.1 (45–82)
      Sex
       Male6 (46.1)
       Female7 (53.9)
      Clinical diagnosis14 atherosclerotic plaques
       Carotid stenosis8 (57.1)
       Aortic aneurysm6 (42.9)
      Medical comorbidities13 patients
      Comorbidities may be associated (ncomorbidities=31).
       Hypertension9 (69.2)
       Dyslipidemia8 (61.5)
       Diabetes2 (15.4)
       Other9 (69.2)
       None3 (23.1)
      Smoking history13 patients
       Smoker1 (7.7)
       Ex-smoker6 (46.2)
       Never smoked6 (46.2)
      Dental profile13 patients/78 sextants
       DMFT31.5 ± 1.4 (27–32)
       Healthy teeth7 (1.7)
       Decayed teeth7 (1.7)
       Filled teeth2 (0.5)
       Missing teeth400 (96.1)
      PSR
       Health sextants0 (0)
       Bleeding sextants0 (0)
       Sextants with dental calculus1 (1.3)
       Sextants with periodontal pockets 4–5 mm1 (1.3)
       Sextants with periodontal pockets >6 mm0 (0)
       Excluded sextants (≤1 dente)76 (97.4)
      - DMFT, decayed, missing, filled teeth; PSR, Periodontal Screening and Recording. Quantitative data expressed by “Mean ± SD (minimum–maximum)”. Qualitative data expressed by “n (%)”.
      a Comorbidities may be associated (ncomorbidities = 31).
      In the present study, histopathologically, all specimens showed features of severe atherosclerotic lesions (Supplemental results). Although there was no morphologic evidence of bacterial colonization in the atherosclerotic plaques, all samples were positive for at least one-studied bacteria. In addition, at least two investigated bacteria were detected in all oral samples, although the majority of the patients were edentulous.
      The presence of oral bacteria in atherosclerotic plaques of edentulous patients may result from recurrent bacteremia that occurred while the patient still had teeth, considering that the process of atherosclerotic plaque formation is a long-term process that can begin in childhood [
      • Zaremba M.
      • Górska R.
      • Suwalski P.
      • Czerniuk M.R.
      • Kowalski J.
      Periodontitis as a risk factor of coronary heart diseases?.
      ]. Another hypothesis is that these bacteria could enter the bloodstream through ulcers or epithelial lesions present in the oral cavity of edentulous patients, adhering to existing atherosclerotic lesions [
      • Zaremba M.
      • Górska R.
      • Suwalski P.
      • Czerniuk M.R.
      • Kowalski J.
      Periodontitis as a risk factor of coronary heart diseases?.
      ].
      S. mutans was identified in all oral samples (100.0%) and atherosclerotic plaques (100.0%) (Supplemental Table 2), which suggests that this bacterium may have been originated from the oral cavity and most likely reached atherosclerotic plaques through bacteremia. The detection of S. mutans at a high frequency in atherosclerotic plaques may be correlated with a high average of lost teeth.
      The role of S. mutans in atherogenesis has been investigated. Several in vitro studies have shown that S. mutans has the ability to adhere to collagen type 1 [
      • Nomura R.
      • Nakano K.
      • Naka S.
      • et al.
      Identification and characterization of a collagen-binding protein, cbm, in Streptococcus mutans.
      ], induce platelet aggregation [
      • Matsumoto-Nakano M.
      • Tsuji M.
      • Inagaki S.
      • et al.
      Contribution of cell surface protein antigen c of streptococcus mutans to platelet aggregation.
      ], invade human endothelial cells [
      • Nagata E.
      • de Toledo A.
      • Oho T.
      Invasion of human aortic endothelial cells by oral viridans group streptococci and induction of inflammatory cytokine production.
      ] and induce increased production of interleukin (IL) 1, IL-6, monocyte chemoattractant protein 1 (MCP-1) and foamy macrophages, which are strongly associated with the pathogenesis of atherosclerosis [
      • Nagata E.
      • de Toledo A.
      • Oho T.
      Invasion of human aortic endothelial cells by oral viridans group streptococci and induction of inflammatory cytokine production.
      ]. Studies using animal models observed that an infection with the invasive strain of S. mutans OMZ175 accelerates the development of atherosclerotic plaques and increases the inflammatory response in an ApoE-null mouse when compared to the control without S. mutans infection [
      • Kesavalu L.
      • Lucas A.R.
      • Verma R.K.
      • et al.
      Increased atherogenesis during Streptococcus mutans infection in ApoE-null mice.
      ]. These results suggest that invasive strains of S. mutans may be related to vascular disease in humans, possibly contributing to the progression of atherosclerotic lesions.
      In the present study, P. intermedia was detected in the oral samples of 12 patients (92.0%). Only one patient (7.1%) presented positivity for this bacterium in the vascular sample, but not in the oral sample. This fact supports the theory that this microorganism may have colonized the atherosclerotic plaque during previous bacteremia while the patient still had teeth.
      Two patients (15.3%) presented positive oral samples for P. gingivalis, and 4 patients (30.7%) for T. denticola. However, these bacteria were not identified in vascular samples. Regarding the frequency of bacteria in atherosclerotic plaques, S. mutans presented a higher prevalence (p < 0.05) compared to the other bacteria (Fisher/X2 exact test with Bonferroni correction) (Supplemental Fig. 1). There was a statistically significant difference regarding the presence of P. gingivalis and T. denticola in the oral cavity compared to the vascular sample (p < 0.05) (Fisher/X2 exact test with Bonferroni correction) (Fig. 1), suggesting that these bacteria have greater difficulty in entering the bloodstream when compared to S. mutans.
      Figure thumbnail gr1
      Fig. 1Percentage distribution of periodontopathic and cariogenic bacteria in oral samples (saliva, supragingival plaque and subgingival plaque) and atherosclerotic plaques. *Statistically significant difference (p < 0.05) between the frequency of Pi in oral samples and in atherosclerotic plaque samples. †Statistically significant difference (p < 0.05) between the frequency of Td in oral samples and in atherosclerotic plaque samples (Fisher exact test or X2 test with Bonferroni correction). Sm, Streptococcus mutans; Pi, Prevotella intermedia; Pg, Porphyromonas gingivalis; Td, Treponema denticola.
      The oral and vascular bacterial profiles of the patients in the present study were distinct because only S. mutans and P. intermedia were detected in atherosclerotic plaques. S. mutans was found at a high frequency in vascular and oral samples, even in edentulous patients, and its presence in atherosclerotic plaques suggests a possible involvement of this pathogen in the disease progression. Therefore, routine dental appointments are important for dentate and edentulous patients, whether they are cardiac patients or patients at risk for atherosclerotic disease, to maintain oral health and to reduce recurrent episodes of bacteremia.
      During the execution of the present study, the authors found difficulty in obtaining the samples, considering that there are currently less invasive and more widely used methods for treating carotid stenosis than endarterectomy, which was a study limitation.

      Acknowledgment

      The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology.
      No outside founding sources supported this work.

      Appendix A. Supplementary data

      References

        • Nakano K.
        • Nemoto H.
        • Nomura R.
        • et al.
        Detection of oral bacteria in cardiovascular specimens.
        Oral Microbiol Immunol. 2009; 24: 64-68
        • Kozarov E.
        • Sweier D.
        • Shelburne C.
        • Progulske-Fox A.
        • Lopatin D.
        Detection of bacterial DNA in atheromatous plaques by quantitative PCR.
        Microbes Infect. 2006; 8: 687-693
        • Nakano K.
        • Inaba H.
        • Nomura R.
        • et al.
        Detection of cariogenic Streptococcus mutans in extirpated heart valve and atheromatous plaque specimens.
        J Clin Microbiol. 2006; 44: 3313-3317
        • Cairo F.
        • Gaeta C.
        • Dorigo W.
        • et al.
        Periodontal pathogens in atheromatous plaques. A controlled clinical and laboratory trial.
        J Periodontal Res. 2004; 39: 442-446
        • Wilson K.
        Preparation of genomic DNA from bacteria.
        Curr Protoc Mol Biol. 2001; : 2-4
        • Zaremba M.
        • Górska R.
        • Suwalski P.
        • Czerniuk M.R.
        • Kowalski J.
        Periodontitis as a risk factor of coronary heart diseases?.
        Adv Med Sci. 2006; 51: 34-39
        • Nomura R.
        • Nakano K.
        • Naka S.
        • et al.
        Identification and characterization of a collagen-binding protein, cbm, in Streptococcus mutans.
        Mol Oral Microbiol. 2012; 27: 308-323
        • Matsumoto-Nakano M.
        • Tsuji M.
        • Inagaki S.
        • et al.
        Contribution of cell surface protein antigen c of streptococcus mutans to platelet aggregation.
        Oral Microbiol Immunol. 2009; 24: 427-430
        • Nagata E.
        • de Toledo A.
        • Oho T.
        Invasion of human aortic endothelial cells by oral viridans group streptococci and induction of inflammatory cytokine production.
        Mol Oral Microbiol. 2011; 26: 78-88
        • Kesavalu L.
        • Lucas A.R.
        • Verma R.K.
        • et al.
        Increased atherogenesis during Streptococcus mutans infection in ApoE-null mice.
        J Dent Res. 2012; 91: 255-260