Atherosclerotic Plaque as Infected Biofilm on Electron Microscopy

by Jeffrey Dach MD

Highlights of the Matters from the Heart ICIM March 2019 Philadelphia Meeting.

At the Recent ICIM Meeting, Dr Thomas Levy gave a great talk on peri-odontal disease as source of origin for infection in atherosclerotic plaque.  He urged all practitioners to have all coronary artery disease patients have 3-D dental xray evaluation looking for underlying dental infection as underlying cause of coronary artery disease.  More of this topic from Dr Levy’s is available in his 2017 book: Hidden Epidemic.by Thomas Levy.

Benefits of the Plant Based Diet

Another speaker at the meeting, Dr Joel Kahn also spoke about the plant based diet.  He also discussed metabolic endotoxemia, ie.”leaky gut” as another source of origin for seeding infection into the atherosclerotic plaque,  thus causing heart disease.  Both Joel Kahn and Kirk Hamilton mentioned the 2017  article by Esselstyn.(40) who conducted a Plant Based Diet study of almost 200 patients with significant coronary artery disease.  Dr Esselstyn says:

“During four years of follow up, 99.4% of the participants who adhered to WFPBN (plant based diet) avoided any major cardiac event including heart attack, stroke, and death, and angina improved or resolved in 93%. Of the 21 non-adherent participants, 13 (62%) experienced an adverse event.”(40)

Kirk Hamilton and Steven Frye, both attending the meeting, speculate that efficacy of the plant based diet may be due to antimicrobial properties of plant roots such as Garlic.  Dr Stephen Fry’s lab has identified soil fungi organisms in atherosclerotic plaque.  Dr Frye mentions that plants root in the soil and are exposed to soil fungi, and over centuries, have developed resistance to soil fungi for their own survival. (Link to Kirk Hamilton podcast with Stephen Fry)

Biological Evidence Supporting Infection-Based Model of Atherogenesis.

Dr Kozorov in 2017 Cardiovascular Research describes two routes for invasion of bacteria  into atherosclerotic plaques (red bugs in diagram below)  .(9-10)  One is by direct invasion across the endothelium or vasa vasorum, the other is indirectly, internalized in macrophages as a “Trojan Horses”.  The bacteria (small red bugs) are carried into the atheroma inside invading macrophages.(see diagram below)

Above diagram: Red bugs are the invading bacteria.  courtesy of : Kozarov, Emil et al.. “View of statins as antimicrobials in cardiovascular risk modification.” Card Res 102.3 (2014): 362-374. Fig 2.  Dr Kozorov says:(10)

“Biological evidence supporting infection-based model of atherogenesis. Both bacteraemia and phagocyte-mediated delivery of bacteria to the site of inflammation are indicated. Bacteraemic organisms are invading the endothelial layer and spreading into vessel wall (left), releasing pro-inflammatory chemokines (such as MCP-1). Blood monocytes (MN) and macrophages (MΦ) are thus activated, prompting their adhesion and diapedesis.  MN, monocyte; MΦ, macrophage with internalized bacteria; EC, endothelial cell; SMC, smooth muscle cell; MCP-1, monocyte chemotactic protein-1; Apoptotic EC, apoptotic endothelial cell releasing intracellular bacteria.” (10)

Dr. Kozorov makes the point that statin drugs are also anti-microbial, and their efficacy is probably due to anti-microbial and anti-inflammatory effects, not due to cholesterol lowering.(9-10)

Scanning Electron Microscopy of Atherosclerotic Plaques Specimens

Above Image Courtesy of : Armingohar, Zahra, et al. “Bacteria and bacterial DNA in atherosclerotic plaque and aneurysmal wall biopsies from patients with and without periodontitis.” Journal of oral microbiology 6.1 (2014): 23408. (1)  Fig. 1 Scanning electron micrographs of bacteria in vascular biopsies from patients with periodontitis. (a) Area of aneurysmal wall with bacteria entangled in meshwork of delicate fibers.  Apparently, division of coccus-shaped bacteria is occurring.

Dr Armingohar in the 2014 Journal of Microbiology studied atherosclerotic plaque and aneurysm wall biopsies in patients with and without periodontitis.(1)   Using scanning electron microscopy, Dr  Armingohar found a rod shaped and spherical shaped forms entangled in a mesh like network compatible with infected biofilm. (see above image)  Some of the microbial life-forms were actively budding.

FISH 16s and 23s Ribosome DNA found in Atheromas

Dr Bernard Lanter’s lab uses fluorescent staining for 16s and 23s ribosome DNA to identify bacterial signatures in atheromas obtained from carotid artery surgical specimens (2)  (See diagram below)  Note two different strains of bacteria (green stain for 16s and red stain for 23s) are located withing the same distribution within the atheroma material.  Dr Lanter found bacterial signatures in diseased tissue, and not in healthy tissue.

Above image courtesy of Bernard Lanter (2): Lanter, B. B., and D. G. Davies. “Propionibacterium acnes Recovered from Atherosclerotic Human Carotid Arteries Undergoes Biofilm Dispersion and Releases Lipolytic and Proteolytic Enzymes in Response to Norepinephrine Challenge In Vitro.” Infection and immunity 83.10 (2015): 3960-3971.(2)

Green fluorescence indicates the presence of the bound eubacterial 16S rRNA gene probe, while red fluorescence indicates the presence of the bound P. acnes 23S rRNA gene probe. Healthy tissue contains no bound probe, while diseased tissue contains bound eubacterial 16S rRNA and P. acnes 23S rRNA gene probes.

Conclusion: The original pathiologists more than 100 years ago considered atherosclerosis to be an infection.  After all these years, modern imaging techniques are proving they were right all along.

Articles with Related Interest:

Coronary Calcium Score, Benefits of Aged Garlic

Heart Book by Jeffry Dach MD

Jeffrey Dach MD

This article is Part Two, for part one click here.

Links and References:

1) Armingohar, Zahra, et al. “Bacteria and bacterial DNA in atherosclerotic plaque and aneurysmal wall biopsies from patients with and without periodontitis.” Journal of oral microbiology 6.1 (2014): 23408.

2) Lanter, B. B., and D. G. Davies. “Propionibacterium acnes Recovered from Atherosclerotic Human Carotid Arteries Undergoes Biofilm Dispersion and Releases Lipolytic and Proteolytic Enzymes in Response to Norepinephrine Challenge In Vitro.” Infection and immunity 83.10 (2015): 3960-3971.

Green fluorescence indicates the presence of the bound eubacterial 16S rRNA gene probe, while red fluorescence indicates the presence of the bound P. acnes 23S rRNA gene probe. Healthy tissue contains no bound probe, while diseased tissue contains bound eubacterial 16S rRNA and P. acnes 23S rRNA gene probes.

3) Lanter, Bernard B., Karin Sauer, and David G. Davies. “Bacteria present in carotid arterial plaques are found as biofilm deposits which may contribute to enhanced risk of plaque rupture.” MBio 5.3 (2014): e01206-14.

4) Schor J and Alschuler L. Bacterial Growth in Arteries Implicated in Heart Attacks Stress, biofilms, and cardiovascular disease  . Natural Medicine Journal August 2014 Vol. 6 Issue 8.

5) Ott SJ, et al. Fungal rDNA signatures in coronary atherosclerotic plaques. Environ Microbiol. 2007;9(12):3035-45.

6) Ott, Stephan J., et al. “Detection of diverse bacterial signatures in atherosclerotic lesions of patients with coronary heart disease.” Circulation 113.7 (2006): 929-937.Detection of diverse bacterial signatures in atherosclerotic lesions of patients with coronary heart disease.

Bacterial infection has been discussed as a potential etiologic factor in the pathophysiology of coronary heart disease (CHD). This study analyzes molecular phylogenies to systematically explore the presence, frequency, and diversity of bacteria in atherosclerotic lesions in patients with CHD.
METHODS AND RESULTS: We investigated 16S rDNA signatures in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients with CHD, control material from postmortem patients (n=15), and heart-beating organ donors (n=11) using clone libraries, denaturating gradient gel analysis, and fluorescence in situ hybridization. Bacterial DNA was found in all CHD patients by conserved PCR but not in control material or in any of the normal/unaffected coronary arteries. Presence of bacteria in atherosclerotic lesions was confirmed by fluorescence in situ hybridization. A high overall bacterial diversity of >50 different species, among them Staphylococcus species, Proteus vulgaris, Klebsiella pneumoniae, and Streptococcus species, was demonstrated in >1500 clones from a combined library and confirmed by denaturating gradient gel analysis. Mean bacterial diversity in atheromas was high, with a score of 12.33+/-3.81 (range, 5 to 22). A specific PCR detected Chlamydia species in 51.5% of CHD patients.
CONCLUSIONS: Detection of a broad variety of molecular signatures in all CHD specimens suggests that diverse bacterial colonization may be more important than a single pathogen. Our observation does not allow us to conclude that bacteria are the causative agent in the etiopathogenesis of CHD. However, bacterial agents could have secondarily colonized atheromatous lesions and could act as an additional factor accelerating disease progression.

7) Fry SE, et al Putative biofilm-forming organisms in the human vasculature: expanded case reports and review of the literature. Phlebological Review 2014; 22(1): 24–37.Biofilm_forming organisms human vasculature Stephen Eugene Fry Phlebological Review 2014

8) Ellis, Jeremy E., et al. “Evidence for polymicrobial communities in explanted vascular filters and atheroma debris.” Molecular and cellular probes 33 (2017): 65-77.

Microbial communities have been implicated in a variety of disease processes and have been intermittently observed in arterial disease; however, no comprehensive unbiased community analysis has been performed. We hypothesize that complex microbial communities may be involved in chronic vascular diseases as well and may be effectively characterized by molecular assays.
OBJECTIVE: The main objective is to survey vascular debris, atheroma, and vascular filters for polymicrobial communities consisting of prokaryotic and eukaryotic microbes, specifically eukaryotic microbes.
METHODS AND RESULTS:  We examined vascular aspirates of atheromatous debris or embolic protection filters in addition to matched peripheral blood samples, from fifteen patients, as well as three cadaveric coronary arteries from two separate patients, for microbial communities. General fluorescence microscopy by Höechst and ethidium bromide DNA stains, prokaryotic and eukaryotic community analysis by Next Generation DNA Sequencing (NGS), and a eukaryotic microbial 9 probe multiplexed quantitative PCR were used to detect and characterize the presence of putative polymicrobial communities. No prokaryotes were detected in peripheral blood; however, in 4 of 9 sequenced filters and in 2 of 7 sequenced atheroma debris samples, prokaryotic populations were identified. By DNA sequencing, eukaryotic microbes were detected in 4 of 15 blood samples, 5 of the 9 sequenced filters, and 3 of the 7 atheroma debris samples. The quantitative multiplex PCR detected sequences consistent with eukaryotic microbes in all 9 analyzed filter samples as well as 5 of the 7 atheroma debris samples. Microscopy reveals putative polymicrobial communities within filters and atheroma debris. The main contributing prokaryotic species in atheroma debris suggest a diverse and novel composition. Additionally, Funneliformis mosseae, an arbuscular mycorrhizal fungus in the Glomeraceae family, was detected in the coronary hard plaque from two patients. Well studied biofilm forming bacteria were not detectable in circulating peripheral blood and were not universally present in atheroma or filters. Analyses of the sequenced eukaryotes are consistent with a diverse of array poorly studied environmental eukaryotes. In summary, out of 15 patients, 6 exhibited molecular evidence of prokaryotes and 14 had molecular evidence of eukaryotic and/or polymicrobial communities in vivo, while 2 post-mortem coronary plaque samples displayed evidence of fungi.
CONCLUSION: Prokaryotes are not consistently observed in atheroma debris or filter samples; however, detection of protozoa and fungi in these samples suggests that they may play a role in arterial vascular disease or atheroma formation.

9)  Kozarov, Emil. “Bacterial invasion of vascular cell types: vascular infectology and atherogenesis.” Future cardiology 8.1 (2012): 123-138.

Cultivation of microorganisms from diseased tissue has been demonstrated,

Biological evidence supporting infection-based model of atherogenesis. Both bacteraemia and phagocyte-mediated delivery of bacteria to the site of inflammation are indicated. Bacteraemic organisms are invading the endothelial layer and spreading into vessel wall (left), releasing pro-inflammatory chemokines (such as MCP-1). Blood monocytes (MN) and macrophages (MΦ) are thus activated, prompting their adhesion and diapedesis. Transmigrating leucocytes (centre) can carry internalized bacteria, thus promoting systemic dissemination of bacteria. In addition, the internalization of bacteria can switch them into uncultivable state (black into grey), while their internalization by phagocytes can reactivate them back to metabolic activity (from grey to black). The growth of atheromas is due in large extent to macrophage-secreted growth factors that induce SMC proliferation. Host cell death (depicted at right) can also lead to pathogen release in the tissue. MN, monocyte; MΦ, macrophage with internalized bacteria; EC, endothelial cell; SMC, smooth muscle cell; MCP-1, monocyte chemotactic protein-1; Apoptotic EC, apoptotic endothelial cell releasing intracellular bacteria.

10) Kozarov, Emil, Teresa Padro, and Lina Badimon. “View of statins as antimicrobials in cardiovascular risk modification.” Cardiovascular research 102.3 (2014): 362-374.

11) Mawhorter SD, Lauer MA. Is atherosclerosis an infectious disease? Cleve Clin J Med. 2001;68(5):449-58.

12) Kuo CC, Shor A, Campbell LA, et al. Demonstration of Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries. J Infect Dis. 1993;167(4):841-9.

13) Koren O, at al. Human oral, gut, and plaque microbiota in patients with atherosclerosis. Proc Natl Acad Sci U S A. 2011;108 Suppl 1:4592-8.

14) Chakravorty, S et al. “Detailed analysis of 16S ribosomal RNA gene segments for diagnosis of pathogenic bacteria.” Journal of microbiological methods 69.2 (2007): 330-339.

15) Brown, J. Mark, and Stanley L. Hazen. “The Gut Microbial Endocrine Organ: Bacterially-Derived Signals Driving Cardiometabolic Diseases.” Annual review of medicine 66 (2015): 343.

“metabolic endotoxemia” because it has been found to be prevalent in many chronic metabolic diseases such as obesity, type II diabetes, and atherosclerosis

LPS in Atheromatoud Plaque

16) Carnevale, Roberto, et al. “Localization of lipopolysaccharide from Escherichia Coli into human atherosclerotic plaque.” Scientific reports 8.1 (2018): 3598.

17) Lehr, Hans-Anton, et al. “Immunopathogenesis of atherosclerosis Endotoxin accelerates atherosclerosis in rabbits on hypercholesterolemic diet.” Circulation 104.8 (2001): 914-920.

LPS challenged Mouse Model

18) Cuaz-Pérolin, Clarisse, et al. “Antiinflammatory and antiatherogenic effects of the NF-κB inhibitor acetyl-11-keto-β-boswellic acid in LPS-challenged ApoE−/− mice.” Arteriosclerosis, thrombosis, and vascular biology 28.2 (2008): 272-277.

Periodontal Disease in Pathogenesis of Atherosclerosis

19) Vojdani, A. “The Role of Periodontal Disease and Other Infections in the Pathogenesis of Atherosclerosis and Systemic Diseases.” Townsend Letter for Doctors and Patients (2000): 52–57.

20) Chhibber-Goel, Jyoti, et al. “Linkages between oral commensal bacteria and atherosclerotic plaques in coronary artery disease patients.” NPJ biofilms and microbiomes 2.1 (2016): 7.

21)  Liljestrand, John M., et al. “Lipopolysaccharide mediator between periodontitis and coronary artery disease Liljestrand J clin periodontology 2017.” Journal of clinical periodontology 44.8 (2017): 784-792.

Transmission electron microscopy identified bacteria in all 9 thombus samples.

22) Pessi, Tanja, et al. “Bacterial signatures in thrombus aspirates of patients with myocardial infarction.” Circulation 127.11 (2013): 1219-1228.

Infectious agents, especially bacteria and their components originating from the oral cavity or respiratory tract, have been suggested to contribute to inflammation in the coronary plaque, leading to rupture and the subsequent development of coronary thrombus. We aimed to measure bacterial DNA in thrombus aspirates of patients with ST-segment-elevation myocardial infarction and to check for a possible association between bacteria findings and oral pathology in the same cohort.
METHODS AND RESULTS:Thrombus aspirates and arterial blood from patients with ST-segment-elevation myocardial infarction undergoing primary percutaneous coronary intervention (n=101; 76% male; mean age, 63.3 years) were analyzed with real-time quantitative polymerase chain reaction with specific primers and probes to detect bacterial DNA from several oral species and Chlamydia pneumoniae. The median value for the total amount of bacterial DNA in thrombi was 16 times higher than that found in their blood samples. Bacterial DNA typical for endodontic infection, mainly oral viridans streptococci, was measured in 78.2% of thrombi, and periodontal pathogens were measured in 34.7%. Bacteria-like structures were detected by transmission electron microscopy in all 9 thrombus samples analyzed; whole bacteria were detected in 3 of 9 cases. Monocyte/macrophage markers for bacteria recognition (CD14) and inflammation (CD68) were detected in thrombi (8 of 8) by immunohistochemistry. Among the subgroup of 30 patients with myocardial infarction examined by panoramic tomography, a significant association between the presence of periapical abscesses and oral viridans streptococci DNA-positive thrombi was found (odds ratio, 13.2; 95% confidence interval, 2.11-82.5; P=0.004).
CONCLUSIONS:

Dental infection and oral bacteria, especially viridans streptococci, may be associated with the development of acute coronary thrombosis.

23) J Periodontol. 2005 Nov;76(11 Suppl):2085-8.
Dental infections and cardiovascular diseases: a review.
Mattila KJ1, Pussinen PJ, Paju S.

Accumulating evidence suggests that chronic infections, such as periodontitis, are associated with increased risk for cardiovascular diseases (CVD). The mechanisms behind the association are not known. Like herpes viruses and Chlamydia pneumoniae, periodontal pathogens cause atherosclerosis in experimental animals and have been found in human atherosclerotic lesions. Higher concentrations of total and low density lipoprotein (LDL) cholesterol and triglycerides and lower concentrations of high density lipoprotein (HDL) cholesterol have been observed in individuals with periodontitis before periodontal treatment. Periodontitis also induces a peripheral inflammatory and immune response, reflected in elevated concentrations of C-reactive protein (CRP) and IgA-class antibodies to periodontal pathogens. The prevalence of CVD seems to be highest in those individuals in whom periodontitis coexists with elevated CRP levels. This may indicate that periodontitis is a CVD risk factor in individuals who react to the infection with a systemic inflammatory and immune response. This may be due to genetic reasons and may also apply to other chronic low-grade infections.

24) Indian J Dent Res. 2010 Apr-Jun;21(2):248-52. 16S rRNA-based detection of oral pathogens in coronary atherosclerotic plaque. 
Mahendra J1, Mahendra L, Kurian VM, Jaishankar K, Mythilli R.

Atherosclerosis develops as a response of the vessel wall to injury. Chronic bacterial infections have been associated with an increased risk for atherosclerosis and coronary artery disease. The ability of oral pathogens to colonize in coronary atheromatous plaque is well known.
AIM:

The aim of this study was to detect the presence of Treponema denticola, Porphyromonas gingivalis and Campylobacter rectus in the subgingival and atherosclerotic plaques of patients with coronary artery disease.
MATERIALS AND METHODS:

Fifty-one patients in the age group of 40-80 years with coronary artery disease were selected for the study. DNA was extracted from the plaque samples. The specific primers for T. denticola, C. rectus and P. gingivalis were used to amplify a part of the 16S rRNA gene by polymerase chain reaction.
STATISTICAL ANALYSIS USED:

Chi-square analysis, correlation coefficient and prevalence percentage of the microorganisms were carried out for the analysis.
RESULTS:

Of the 51 patients, T. denticola, C. rectus and P. gingivalis were detected in 49.01%, 21.51% and 45.10% of the atherosclerotic plaque samples.
CONCLUSIONS:

Our study revealed the presence of bacterial DNA of the oral pathogenic microorganisms in coronary atherosclerotic plaques. The presence of the bacterial DNA in the coronary atherosclerotic plaques in significant proportion may suggest the possible relationship between periodontal bacterial infection and genesis of coronary atherosclerosis.

25) J Periodontol. 2000 Oct;71(10):1554-60.
Identification of periodontal pathogens in atheromatous plaques.
Haraszthy VI1, Zambon JJ, Trevisan M, Zeid M, Genco RJ.

Recent studies suggest that chronic infections including those associated with periodontitis increase the risk for coronary vascular disease (CVD) and stroke. We hypothesize that oral microorganisms including periodontal bacterial pathogens enter the blood stream during transient bacteremias where they may play a role in the development and progression of atherosclerosis leading to CVD.
METHODS: To test this hypothesis, 50 human specimens obtained during carotid endarterectomy were examined for the presence of Chlamydia pneumoniae, human cytomegalovirus, and bacterial 16S ribosomal RNA using specific oligonucleotide primers in polymerase chain reaction (PCR) assays. Approximately 100 ng of chromosomal DNA was extracted from each specimen and then amplified using standard conditions (30 cycles of 30 seconds at 95 degrees C, 30 seconds at 55 degrees C, and 30 seconds at 72 degrees C). Bacterial 16S rDNA was amplified using 2 synthetic oligonucleotide primers specific for eubacteria. The PCR product generated with the eubacterial primers was transferred to a charged nylon membrane and probed with digoxigenin-labeled synthetic oligonucleotides specific for Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Porphyromonas gingivalis, and Prevotella intermedia.
RESULTS: Eighty percent of the 50 endarterectomy specimens were positive in 1 or more of the PCR assays. Thirty-eight percent were positive for HCMV and 18% percent were positive for C. pneumoniae. PCR assays for bacterial 16S rDNA also indicated the presence of bacteria in 72% of the surgical specimens. Subsequent hybridization of the bacterial 16S rDNA positive specimens with species-specific oligonucleotide probes revealed that 44% of the 50 atheromas were positive for at least one of the target periodontal pathogens. Thirty percent of the surgical specimens were positive for B. forsythus, 26% were positive for P. gingivalis, 18% were positive for A. actinomycetemcomitans, and 14% were positive for P. intermedia. In the surgical specimens positive for periodontal pathogens, more than 1 species was most often detected. Thirteen (59%) of the 22 periodontal pathogen-positive surgical specimens were positive for 2 or more of the target species.
CONCLUSIONS:  Periodontal pathogens are present in atherosclerotic plaques where, like other infectious microorganisms such as C. pneumoniae, they may play a role in the development and progression of atherosclerosis leading to coronary vascular disease and other clinical sequelae.

Infection Found in Cerebral Aneurysms

26) J Neurol Neurosurg Psychiatry. 2013 Nov;84(11):1214-8.
The connection between ruptured cerebral aneurysms and odontogenic bacteria.  Pyysalo MJ1, Pyysalo LM, Pessi T, Karhunen PJ, Öhman JE.

Patients with ruptured saccular intracranial aneurysms have excess long-term mortality due to cerebrovascular and cardiovascular diseases compared with general population. Chronic inflammation is detected in ruptured intracranial aneurysms, abdominal aortic aneurysms and coronary artery plaques. Bacterial infections have been suggested to have a role in the aetiology of atherosclerosis. Bacteria have been detected both in abdominal and coronary arteries but their presence in intracranial aneurysms has not yet been properly studied.
OBJECTIVE:  The aim of this preliminary study was to assess the presence of oral and pharyngeal bacterial genome in ruptured intracranial aneurysms and to ascertain if dental infection is a previously unknown risk factor for subarachnoid haemorrhage.
METHODS:  A total of 36 ruptured aneurysm specimens were obtained perioperatively in aneurysm clipping operations (n=29) and by autopsy (n=7). Aneurysmal sac tissue was analysed by real time quantitative PCR with specific primers and probes to detect bacterial DNA from several oral species. Immunohistochemical staining for bacterial receptors (CD14 and toll-like receptor-2 (TLR-2)) was performed from four autopsy cases.
RESULTS:  Bacterial DNA was detected in 21/36 (58%) of specimens. A third of the positive samples contained DNA from both endodontic and periodontal bacteria. DNA from endodontic bacteria were detected in 20/36 (56%) and from periodontal bacteria in 17/36 (47%) of samples. Bacterial DNA of the Streptococcus mitis group was found to be most common. Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum and Treponema denticola were the three most common periodontal pathogens. The highly intensive staining of CD14 and TLR-2 in ruptured aneurysms was observed.
CONCLUSIONS:  This is the first report showing evidence that dental infection could be a part of pathophysiology in intracranial aneurysm disease.

27) Acta Odontol Scand. 2016;74(4):315-20. Bacterial DNA findings in ruptured and unruptured intracranial aneurysms.
Pyysalo MJ1,2, Pyysalo LM3, Pessi T4, Karhunen PJ4,5, Lehtimäki T6, Oksala N6,7, Öhman JE3.

Chronic inflammation has earlier been detected in ruptured intracranial aneurysms. A previous study detected both dental bacterial DNA and bacterial-driven inflammation in ruptured intracranial aneurysm walls. The aim of this study was to compare the presence of oral and pharyngeal bacterial DNA in ruptured and unruptured intracranial aneurysms. The hypothesis was that oral bacterial DNA findings would be more common and the amount of bacterial DNA would be higher in ruptured aneurysm walls than in unruptured aneurysm walls.
MATERIALS AND METHODS:  A total of 70 ruptured (n = 42) and unruptured (n = 28) intracranial aneurysm specimens were obtained perioperatively in aneurysm clipping operations. Aneurysmal sac tissue was analysed using a real-time quantitative polymerase chain reaction to detect bacterial DNA from several oral species. Both histologically non-atherosclerotic healthy vessel wall obtained from cardiac by-pass operations (LITA) and arterial blood samples obtained from each aneurysm patient were used as control samples.
RESULTS:  Bacterial DNA was detected in 49/70 (70%) of the specimens. A total of 29/42 (69%) of the ruptured and 20/28 (71%) of the unruptured aneurysm samples contained bacterial DNA of oral origin. Both ruptured and unruptured aneurysm tissue samples contained significantly more bacterial DNA than the LITA control samples (p-values 0.003 and 0.001, respectively). There was no significant difference in the amount of bacterial DNA between the ruptured and unruptured samples.
CONCLUSION:  Dental bacterial DNA can be found using a quantitative polymerase chain reaction in both ruptured and unruptured aneurysm walls, suggesting that bacterial DNA plays a role in the pathogenesis of cerebral aneurysms in general, rather than only in ruptured aneurysms.

Pericardial Fluid

28) Louhelainen, Anne-Mari, et al. “Oral bacterial DNA findings in pericardial fluid.” Journal of oral microbiology 6.1 (2014): 25835.

We recently reported that large amounts of oral bacterial DNA can be found in thrombus aspirates of myocardial infarction patients. Some case reports describe bacterial findings in pericardial fluid, mostly done with conventional culturing and a few with PCR; in purulent pericarditis, nevertheless, bacterial PCR has not been used as a diagnostic method before.
OBJECTIVE:  To find out whether bacterial DNA can be measured in the pericardial fluid and if it correlates with pathologic-anatomic findings linked to cardiovascular diseases.
METHODS:  Twenty-two pericardial aspirates were collected aseptically prior to forensic autopsy at Tampere University Hospital during 2009-2010. Of the autopsies, 10 (45.5%) were free of coronary artery disease (CAD), 7 (31.8%) had mild and 5 (22.7%) had severe CAD. Bacterial DNA amounts were determined using real-time quantitative PCR with specific primers and probes for all bacterial strains associated with endodontic disease (Streptococcus mitis group, Streptococcus anginosus group, Staphylococcus aureus/Staphylococcus epidermidis, Prevotella intermedia, Parvimonas micra) and periodontal disease (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatus, and Dialister pneumosintes).
RESULTS:  Of 22 cases, 14 (63.6%) were positive for endodontic and 8 (36.4%) for periodontal-disease-associated bacteria. Only one case was positive for bacterial culturing. There was a statistically significant association between the relative amount of bacterial DNA in the pericardial fluid and the severity of CAD (p=0.035).
CONCLUSIONS:  Oral bacterial DNA was detectable in pericardial fluid and an association between the severity of CAD and the total amount of bacterial DNA in pericardial fluid was found, suggesting that this kind of measurement might be useful for clinical purposes.

bacteria colonize Aortic Aneurysms

A wide variety of bacteria, including oral bacteria, was found to colonize aortic aneurysms and may play a role in their development.

29) da Silva, Rafael Marques, et al. “Bacterial diversity in aortic aneurysms determined by 16S ribosomal RNA gene analysis.” Journal of vascular surgery 44.5 (2006): 1055-1060.

A wide variety of bacteria, including oral bacteria, was found to colonize aortic aneurysms and may play a role in their development.
Aortic aneurysms are common vascular conditions that cause considerable morbidity and mortality. Understanding of the mechanisms involved in the pathogenesis of the condition remains limited. Recently, infection has been suggested as possible contributor in the development of the disease. The aim of the present study was to examine aortic aneurysms for the presence of bacterial DNA using polymerase chain reaction (PCR) targeting the 16S ribosomal RNA (rRNA) gene, followed by cloning and sequencing.

METHODS:Universal eubacterial primers were used to amplify 16S rRNA bacterial genes in 10 specimens from arterial walls of aortic aneurysms. Subsequently, PCR amplicons were cloned into Escherichia coli and sequencing of the cloned inserts was used to determine species identity or closest relatives by comparison with known sequences in GenBank.
RESULTS:Sequences of Stenotrophomonas spp., including S. maltophilia (formerly Pseudomonas homology group V) were detected in six aneurysm samples. Propionibacterium acnes was identified in five samples, and Brevundimonas diminuta (formerly P. diminuta) in four samples. Other species previously assigned to the Pseudomonas genus such as Comamonas testosteroni, Delftia acidovorans, Burkholderia cepacia, Herbaspirillum sp., and Acidovorax sp. were also detected. Some clones fell into other environmental species, including Methylobacterium sp. and Bradyrhizobium elkanii, and others represented bacteria that have not yet been cultivated. DNA sequences from oral bacteria, including Streptococcus sanguinis, Tannerella forsythia, and Leptotrichia buccalis were detected. Sequences from Prevotella melaninogenica and Lactobacillus delbrueckii, which are commonly found in both mouth and gastrointestinal tract, were also detected. Additional species included Dermacoccus spp. and Corynebacterium vitaeruminis.
CONCLUSIONS:A wide variety of bacteria, including oral bacteria, was found to colonize aortic aneurysms and may play a role in their development. Several of these microorganisms have not yet been cultivated.
CLINICAL RELEVANCE:Although Chlamydophila pneumoniae has been detected in aneurysmal walls, its exact role in the condition remains inconclusive. Overall, there is scarce information about the role of microorganisms in aneurysmal disease. In the present study, we used molecular genetics to detect a diversity of bacteria in arterial walls of aortic aneurysms. The presence of multiple microorganisms in aneurysmal disease may have implications for chemoprophylaxis and antibiotic treatment if directed only at C.pneumoniae.

30) Renko, Jaana, et al. “Bacterial signatures in atherosclerotic lesions represent human commensals and pathogens.” Atherosclerosis 201.1 (2008): 192-197.we defined bacterial DNA signatures in surgically removed sterile abdominal aorta samples of patients with aortic atherosclerosis.

Renko, Jaana, et al. “Bacterial DNA signatures in carotid atherosclerosis represent both commensals and pathogens of skin origin.” European Journal of Dermatology 23.1 (2013): 53-58.nearly all (94%) of the sequences were associated with the human skin microbiome.

31) Paraskevas, K. I., D. P. Mikhailidis, and A. D. Giannoukas.Periodontitis and abdominal aortic aneurysms: a random association or a pathogenetic link?International angiology: a journal of the International Union of Angiology 28.6 (2009): 431.

A number of micro-organisms have been implicated in the development/progression of abdominal aortic aneurysms (AAAs), thus suggesting an infective theory of AAA pathogenesis. Periodontitis may be involved in the development of AAAs by means of introduction of subgingival plaque periodontal bacteria into the bloodstream and degeneration of the aortic wall. A different theory supports that the findings of periodontal pathogens in AAA biopsies are a secondary phenomenon with transient bacteremia leading to invasion of already formed AAAs. It is not yet clear whether the periodontopathic bacteria accelerate the growth/weakening of the aortic wall or whether they are secondary colonizers of AAAs. Clarification of the association between periodontal disease and AAAs in large-scale studies holds implications for a role for chemoprophylaxis/antibiotic treatment in the management of AAAs.

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Leaky Gut – Endotoxemia

32) Fukui, H. ” Endotoxin and Other Microbial Translocation Markers in the Blood: A Clue to Understand Leaky Gut Syndrome” Cell Mol Med 2 (2016): 3.  Endotoxin and microbial translocation markers in blood leaky gut syndrome Fukui Cell Mol Med 2016

Biofilm in Atherosclerotic Plaques

Diabetic Amputations

33) Snow, D. E., et al. “The presence of biofilm structures in atherosclerotic plaques of arteries from legs amputated as a complication of diabetic foot ulcers.” Journal of wound care 25.Sup2 (2016): S16-S22.Bacterial Biofilm in atherosclerotic plaques of leg arteries amputated diabetic foot ulcers Snow J of wound care 2016

Atherosclerosis, rather than microcirculatory impairment caused by endothelial cell dysfunction, is the main driver of circulatory compromise in patients with diabetic limbs. The presence of atherosclerotic plaque at the trifurcation is a significant contributor to amputation of diabetic legs. The presence of bacteria and other microorganisms in atherosclerotic plaque has long been known, however, the cause of chronic inflammation and the role of bacteria/viruses in atherosclerosis have not been studied in detail. The objective of this study was to clarify the cause of the chronic inflammation within atherosclerotic plaques, and determine if any bacteria and/or viruses are involved in the inflammatory pathway.
METHOD:This study uses fluorescence microscopy and fluorescence in-situ hybridisation (FISH) to identify components of biofilm in atherosclerotic arteries. These tools are also used to identify individual bacteria, and determine the architectural spatial location within the atherosclerotic plaque where the bacteria can be found.
RESULTS:The results indicate that the presence of biofilms in grossly involved arteries may be an important factor in chronic inflammatory pathways of atherosclerotic progression, in the amputated limbs of patients with diabetic foot ulcers and vascular disease.
CONCLUSION:While the presence of bacterial biofilm structures in atherosclerotic plaque does not prove that biofilm is the proximate cause of atherosclerosis, it could contribute to the persistent inflammation associated with it. Second, the synergistic relationship between the atherosclerotic infection and the diabetic foot ulcer may ultimately contribute to higher amputation rates in diabetics.

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Pseudomonas in coronary Thrombi

34) Hansen, Gorm Mørk, et al. “Pseudomonas aeruginosa Microcolonies in Coronary Thrombi from Patients with ST-Segment Elevation Myocardial Infarction.” PloS one 11.12 (2016): e0168771.
Chronic infection is associated with an increased risk of atherothrombotic disease and direct bacterial infection of arteries has been suggested to contribute to the development of unstable atherosclerotic plaques. In this study, we examined coronary thrombi obtained in vivo from patients with ST-segment elevation myocardial infarction (STEMI) for the presence of bacterial DNA and bacteria.

Aspirated coronary thrombi from 22 patients with STEMI were collected during primary percutaneous coronary intervention and arterial blood control samples were drawn from radial or femoral artery sheaths.

Analyses were performed using 16S polymerase chain reaction and with next-generation sequencing to determine bacterial taxonomic classification. In selected thrombi with the highest relative abundance of Pseudomonas aeruginosa DNA, peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) with universal and species specific probes was performed to visualize bacteria within thrombi. From the taxonomic analysis we identified a total of 55 different bacterial species.

DNA from Pseudomonas aeruginosa represented the only species that was significantly associated with either thrombi or blood and was >30 times more abundant in thrombi than in arterial blood (p<0.0001).

Whole and intact bacteria present as biofilm microcolonies were detected in selected thrombi using universal and P. aeruginosa-specific PNA-FISH probes. P. aeruginosa and vascular biofilm infection in culprit lesions may play a role in STEMI, but causal relationships remain to be determined.

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Several samples show bacterial DNA far exceeding what would be expected by contamination, suggesting the bacteria may be propagating in the plaque.

35) Wolcott RD, Wolcott JJ, Palacio C, Rodriguez S (2012) A Possible Role of Bacterial Biofilm in the Pathogenesis of Atherosclerosis. J Bacteriol Parasitol 3:127. Bacterial Biofilm in Pathogenesis of Atherosclerosis Wolcott RD J Bact Parasit 2012

Multiple culture and molecular based studies have established the presence of bacteria in atherosclerotic plaques. Although bacteria are present within the plaque, there is no clear understanding or putative pathway as to what part bacteria might play, if any, in the pathogenesis of atherosclerosis. The current models for the pathogenesis of atherosclerotic plaque suggest that persistent infl ammation is an important factor; however, the possible sources for this sustained infl ammation are limited. The concept of biofilm infection, “a new paradigm of bacterial pathogenesis,” is introduced to show that bacteria, organized into a biofilm phenotype mode of growth, produces a sustained hyper-inflammatory host niche. Biofi lm produces an oxidative environment in a host infection. Samples of plaque from 10 patients were examined to compare 16S rDNA to 18S rDNA. Also 4 samples were evaluated in 2 separate locations to evaluate the homogeneity of bacteria within the sample. The 16S rDNA was also sequenced to identify the microorganisms present and their relative contribution to the sample. Several samples demonstrated large amounts of bacterial DNA. The spatial arrangement of bacterial DNA showed a very heterogeneous distribution of bacteria in the plaque. A heat map data analysis shows that for samples that were evaluated in 2 locations the bacteria identifi ed closely correlated. For all the samples combined, the predominant microbial species identifi ed have often been associated with the oral cavity. Several samples show bacterial DNA far exceeding what would be expected by contamination, suggesting the bacteria may be propagating in the plaque. If bacteria are propagating within the plaque, this would most likely be a biofi lm phenotype mode of growth. Biofi lm is known to produce a hyperinfl ammatory response in host environments, and therefore is a candidate for being the “engine” for the persistent infl ammation necessary for the pathogenesis of atherosclerosis.

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Peri-Odontal Oral Infection

36) Chukkapalli, Sasanka S., et al. “Polymicrobial oral infection with four periodontal bacteria orchestrates a distinct inflammatory response and atherosclerosis in ApoEnull mice.” PLoS One 10.11 (2015): e0143291.
Periodontal disease (PD) develops from a synergy of complex subgingival oral microbiome, and is linked to systemic inflammatory atherosclerotic vascular disease (ASVD). To investigate how a polybacterial microbiome infection influences atherosclerotic plaque progression, we infected the oral cavity of ApoEnull mice with a polybacterial consortium of 4 well-characterized periodontal pathogens, Porphyromonas gingivalis, Treponema denticola, Tannerealla forsythia and Fusobacterium nucleatum, that have been identified in human atherosclerotic plaque by DNA screening. We assessed periodontal disease characteristics, hematogenous dissemination of bacteria, peripheral T cell response, serum inflammatory cytokines, atherosclerosis risk factors, atherosclerotic plaque development, and alteration of aortic gene expression. Polybacterial infections have established gingival colonization in ApoEnull hyperlipidemic mice and displayed invasive characteristics with hematogenous dissemination into cardiovascular tissues such as the heart and aorta. Polybacterial infection induced significantly higher levels of serum risk factors oxidized LDL (p < 0.05), nitric oxide (p < 0.01), altered lipid profiles (cholesterol, triglycerides, Chylomicrons, VLDL) (p < 0.05) as well as accelerated aortic plaque formation in ApoEnull mice (p < 0.05). Periodontal microbiome infection is associated with significant decreases in Apoa1, Apob, Birc3, Fga, FgB genes that are associated with atherosclerosis. Periodontal infection for 12 weeks had modified levels of inflammatory molecules, with decreased Fas ligand, IL-13, SDF-1 and increased chemokine RANTES. In contrast, 24 weeks of infection induced new changes in other inflammatory molecules with reduced KC, MCSF, enhancing GM-CSF, IFNγ, IL-1β, IL-13, IL-4, IL-13, lymphotactin, RANTES, and also an increase in select inflammatory molecules. This study demonstrates unique differences in the host immune response to a polybacterial periodontal infection with atherosclerotic lesion progression in a mouse model.

Mouse Model of periodontal bacteria spreading to atherosclerotic plaques

37) Chukkapalli, Sasanka S., et al. “Sequential colonization of periodontal pathogens in induction of periodontal disease and atherosclerosis in LDLRnull mice.” Pathogens and disease 75.1 (2017).

Periodontal disease (PD) and atherosclerotic vascular disease (ASVD) are both chronic inflammatory diseases with a polymicrobial etiology and have been epidemiologically associated. The purpose is to examine whether periodontal bacteria that infect the periodontium can also infect vascular tissues and enhance pre-existing early aortic atherosclerotic lesions in LDLR^null mice. Mice were orally infected with intermediate bacterial colonizer Fusobacterium nucleatum for the first 12 weeks followed by late bacterial colonizers (Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia) for the remaining 12 weeks mimicking the human oral microbiota ecological colonization. Genomic DNA from all four bacterial was detected in gingival plaque by PCR, consistently demonstrating infection of mouse gingival surfaces. Infected mice had significant levels of IgG and IgM antibodies, alveolar bone resorption, and showed apical migration of junctional epithelium revealing the induction of PD. These results support the ability of oral bacteria to cause PD in mice. Detection of bacterial genomic DNA in systemic organs indicates hematogenous dissemination from the gingival pockets.

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plaque is infected Biofilm

38) Jonsson, Annika Lindskog, et al. “Bacterial profile in human atherosclerotic plaques.” Atherosclerosis 263 (2017): 177-183.

We confirmed the presence of bacterial DNA in the atherosclerotic plaque by qPCR analysis of the 16S rRNA gene but observed no difference (n.s.) in the amount between either asymptomatic and symptomatic patients or different plaque regions A, B and C. Unweighted UniFrac distance metric analysis revealed no distinct clustering of samples by patient group or plaque region. Operational taxonomic units (OTUs) from 5 different phyla were identified, with the majority of the OTUs belonging to Proteobacteria (48.3%) and Actinobacteria (40.2%). There was no difference between asymptomatic and symptomatic patients, or plaque regions, when analyzing the origin of DNA at phylum, family or OTU level (n.s.).
Conclusions    There were no major differences in bacterial DNA amount or microbial composition between plaques from asymptomatic and symptomatic patients or between different plaque regions, suggesting that other factors are more important in determining plaque vulnerability.

intestinal bacteria in plaques may original from the intestine.

39) Li, Chuanwei, et al. “Zonulin regulates intestinal permeability and facilitates enteric bacteria permeation in coronary artery disease.” Scientific reports 6 (2016): 29142.

The detection of intestinal bacterial by 16S rRNA gene amplification in both the blood sample and atherosclerotic plaques supports the notion that intestinal bacteria in plaques may original from the intestine.

Given the fact that zonulin is significantly elevated in atherosclerosis and increase intestinal IP, it’s possible that targeting zonulin using monoclonal antibody or inhibitors maybe a provocatively new way to move forward in CAD preventi and treatment.

Plant Based Diet Prevents MI

40) Esselstyn, Caldwell B. “A plant-based diet and coronary artery disease: a mandate for effective therapy.” Journal of geriatric cardiology: JGC 14.5 (2017): 317.

In 2014, we conducted a second larger study of 198 patients with significant CAD.[9] Of these patients, 119 had undergone a prior coronary intervention with stents or bypass surgery, and 44 had a previous heart attack. There were multiple comorbidities including hypercholesterolemia, hypertension, obesity, and diabetes. During four years of follow up, 99.4% of the participants who adhered to WFPBN avoided any major cardiac event including heart attack, stroke, and death, and angina improved or resolved in 93%. Of the 21 non-adherent participants, 13 (62%) experienced an adverse event. When comparing these results to the well-known COURAGE,[13] and Lyon Diet Heart Study,[14] which consisted of conventionally treated participants, there is beyond a 30-fold difference in major cardiovascular events favoring WFPBN.

Rheumatoid Arthritis

41) Curran, Samuel A., et al. “Bacteria in the adventitia of cardiovascular disease patients with and without rheumatoid arthritis.” PloS one 9.5 (2014): e98627.

The incidence of atherosclerosis is significantly increased in rheumatoid arthritis (RA). Infection is one factor that may be involved in the pathogenesis of both diseases. The cause of RA and atherosclerosis is unknown, and infection is one of the factors that may be involved in the pathogenesis of both diseases. The aims of this study were to identify bacteria in the aortic adventitia of patients with cardiovascular disease (CVD) in the presence and absence of RA, and to determine the effect of identified candidate pathogens on Toll-like receptor (TLR)-dependent signalling and the proinflammatory response. The aortic adventitia of 11 CVD patients with RA (RA+CVD) and 11 CVD patients without RA (CVD) were collected during coronary artery bypass graft surgery. Bacteria were detected in four samples from CVD patients and three samples from RA+CVD patients and identified by 16S rRNA gene sequencing. Methylobacterium oryzae was identified in all three RA+CVD samples, representing 44.1% of the bacterial flora. The effect of M. oryzae on TLR-dependent signalling was determined by transfection of HEK-293 cells. Although mild TLR2 signalling was observed, TLR4 was insensitive to M. oryzae. Human primary macrophages were infected with M. oryzae, and a TLDA qPCR array targeting 90 genes involved in inflammation and immune regulation was used to profile the transcriptional response. A significant proinflammatory response was observed, with many of the up-regulated genes encoding proinflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α) and chemokines (CCR7, IL-8). The aortic adventitia of CVD patients contains a wide range of bacterial species, and the bacterial flora is significantly less diverse in RA+CVD than CVD patients. M. oryzae may stimulate an proinflammatory response that may aggravate and perpetuate the pathological processes underlying atherosclerosis in RA patients.

Published on March 25th, 2019 by
Jeffrey Dach MD

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