Prevalence of Microorganisms in Atherosclerotic Plaques of Coronary Arteries: A Systematic Review and Meta-Analysis

Background In this systematic review and meta-analysis, the existence of pathogens in atherosclerotic plaques of coronary arteries was investigated in coronary arteries diseases (CAD) patients. Methods This study was designed and implemented up to 31 August 2020. The findings present according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) checklist. Two independent reviewers (I.RJ and S.H) performed a comprehensive search on four different English databases including PubMed, ISI, Scopus, and Embase. In order to assess the quality of the articles, a checklist prepared by The Joanna Briggs Institute (JBI) was used. Results Finally, 44 studies were selected. The prevalence of different microorganisms in coronary arteries were as follows: Aggregatibacter actinomycetemcomitans (46.2%), Campylobacter rectus (43.0%), Chlamydia pneumonia (42.8%), Cytomegalovirus (29.1%), Helicobacter pylori (18.9%), Herpes simplex virus type 1 (5.9%), Porphyromonas gingivalis (42.6%), Prevotella intermedia (47.6%), Tannerella forsythia (43.7%), and Treponema denticola (32.9%). Conclusion Based on the result of this meta-analysis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans are the most common microorganisms in atherosclerotic plaques of coronary arteries and may have an important role in the development of atherosclerosis.


Introduction
Cardiovascular disease (CVD) is increasingly challenging people's health, irrespective of age, gender, and race. Coronary artery disease (CAD) is the main culprit in the growing burden of CVD [1]. CAD clinically appeared as the focal thickening of the intima layer due to the formation of atheroma [1]. Atheroma is the main content of atherosclerotic plaque constituting macrophages, cholesterol, smooth muscle cells, and dystrophic calcifcation [2]. Association of atherosclerosis with both innate and adaptive immunity is evidenced [3]. Chronic infammation, endothelial dysfunction, and lipid accumulation in the vasculature are hallmarks of atherosclerosis [4].
Formation and development of atherosclerosis are feasible in the absence of traditional risk factors [4].
Surprisingly, nearly half of the patients with CVD are free from known cardiovascular risk factors such as hypercholesterolemia, hypertension, smoking, and obesity [5]. In order to reduce related morbidity and mortality, precise identifcation of all underlying reasons is of paramount importance.
Infections involve, at least in part, in CAD initiation and development [1]. Atherosclerosis may be originated from bacterial infection in terms of microbial symbiosis and infammatory stimulus [6,7]. Microbial agents contribute to the atherosclerosis process directly by infecting the vascular cells or indirectly by activation of infammatory cytokines [4]. In this systematic review and meta-analysis, the existence of pathogens in atherosclerotic plaques of coronary arteries was investigated in CAD patients.

Study Design.
Te present study is a systematic review and meta-analysis investigating the existence of microorganisms isolated from atherosclerotic plaques of coronary arteries. Tis study was designed and implemented up to 31 August 2020. Te fndings present according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) checklist.

Information Sources and Search
Strategy. Two independent reviewers (IRJ and SH) performed a comprehensive search on four diferent English databases including PubMed, ISI, Scopus, and Embase. Specifc search strategies were designed and used for each database. Te included keywords were previous isolated bacterial species (

Eligibility Criteria.
Articles were imported into the EndNote software (version X7). Duplicate ones were automatically deleted from the dataset. Eligibility criteria were original studies on human samples published in English reporting microbial footprints in atherosclerotic plaques of coronary arteries. Studies published in other languages other than English, papers without full text, review articles, and articles presented in conferences as abstracts were excluded.

Quality Assessment.
In order to assess the quality of the articles, a checklist prepared by Te Joanna Briggs Institute (JBI) was used. Te purpose of this appraisal is to assess the methodological quality of a study and to determine the extent to which a study has addressed the possibility of bias in its design, conduct, and analysis. All papers were evaluated on the basis of data relevance and methodological rigor.

2.5.
Screening of the Articles. Primary searching of titles and abstracts was done by IRJ and ZE. Tey separately extracted data as well as evaluation of the articles' quality control. In case of ambiguity in one article, the principal investigator issued the fnal decision.

Data Extraction Form.
A predefned checklist was prepared in order to extract data from all eligible articles.
Tis checklist contained the author name, publishing year, country of origin, method of microorganism detection, and prevalence of each isolated microorganism.

Statistical Analysis.
Heterogeneity between studies and their combination were checked by the Cochran test and I 2 parameter, respectively. In the case of heterogeneity, a random efect model with the inverse variance method was used. Otherwise, the fxed efect model was used. All the analyses were performed by CMA statistical software (ver. 2).

Description of Search.
Following searching of four defned databases (PubMed, ISI, SCOPUS, and Embase), 24343 articles were found which was decreased to 11382 after duplicate deletion. After screening their title and abstracts, 10862 articles were omitted owing to irrelevance to the study aim. Te whole text of 520 papers was read and 44 were used for fnal analysis. It should be noted that references of the enrolled articles were screened as well to include eligible articles. Te fowchart of the enrolled articles was depicted in Figure 1. Te characteristics of included studies were demonstrated in Table 1.

Characteristics of the Included Studies.
Based on geographical distribution, seven studies were conducted in Iran, fve studies in Poland, fve studies in India, three studies in Japan, four studies in Germany, four studies in Brazil, two studies in Korea, two studies in Turkey, two studies in the UK, and one study in Finland, Netherlands, Spain, Serbia, Belgium, France, Canada, Japan and USA, USA, and Denmark.

Discussion
At present, CAD is a worldwide cause of hospitalization and mortality [6]. Beside well-established risk factors such as genetic susceptibility, hypertension, hypercholesterolemia, and smoking, the risk of atherosclerotic vascular disease is increased by microbial infection [8]. For the frst time, the association of microbial infection with atherosclerosis was demonstrated by Marek's disease virus. Tis virus induced atherosclerosis in chickens [9]. To substantiate, vaccination against this virus signifcantly protected chickens from atherosclerosis [10]. Te human microbiome which mainly accumulates in the gut and oral cavity has an outstanding role to human health. Pathogens were found in thrombotic samples of MI patients [11]. Other than CVD, this huge microbial population is also involved in cardiovascular risk factors such as obesity and diabetes [12][13][14][15].
Te potential of pathogens in promoting chronic disorders even in remote organs like colon cancers and atherosclerosis has been evidenced [16]. Tat is why chronic periodontal infection is in close relation to acute myocardial infarction (MI) and CAD [17,18]. It may be interesting if you know that more than 275 oral bacterial species could enter the bloodstream through injured capillaries [19,20]. Oral bioflm is in close proximity to the periodontal vasculature, and this makes the entrance and spread of bacteria feasible even to remote organs like the heart [21]. Indeed, in the case of infammation (periodontitis or gingivitis), bacterial entrance is further smoothed due to increased permeability of the adjacent vessels [22]. Following bacterial access to the bloodstream, they could be localized in different parts of the human body, especially in sites with pathologic changes [23,24]. In one study, subgingival plaques and diferent blood vessels of patients with atherosclerosis and periodontitis were sought in order to determine the frequency of periodontal bacteria. Findings revealed that the most and the least prevalent bacteria were Tannerella forsythensis and Treponema denticola, respectively. Also, the prevalence of A. actinomycetemcomitans and Prevotella intermedia were signifcantly diferent between subjects below and over 60 years of age [25].
Te role of certain microbial infections such as C. pneumoniae, P. gingivalis, A. actinomycetemcomitans, H. pylori, infuenza virus, and cytomegalovirus has been confrmed in the development of atherosclerosis in animal models [4]. Infection of vascular cells, detection of the microbes within the atherosclerotic plaque, and the development of atherosclerotic lesions after microbial infection in animal models reinforce the direct association of infection with atherosclerosis. On the other hand, indirect efects represent the emergence of cytokines and acute phase proteins followed by infection at nonvascular sites [4].
Te immune system is a major part of atherosclerosis progression. Microbial infection instigates immune responses [65]. Production of antibody is emerged after bacterial infections. Tis immune reaction increases the expression of infammatory mediators, which in turn involves in the development of coronary thrombosis [65]. Also, microbial infections, either bacterial or viral, activate vessel-associated leucocytes [65]. Te binding of bacterialassociated molecular patterns to Toll-like receptors triggers a  Evidence-Based Complementary and Alternative Medicine signaling pathway, which upregulates some genes with a key role in atherogenesis (adhesion molecules in endothelial cells and infammatory mediators in immune cells) [66]. Some bacteria like P. gingivalis accelerate atherosclerosis through enhancing the T17 responses [3]. A specifc immune response and aortic infammation are raised with chronic oral P. gingivalis infection [67]. Activation of platelets by bacteria results in thrombosis formation [1,68]. Some bacteria like streptococci are endothelial-adhesive. Tis facilitates embedding to the   Evidence-Based Complementary and Alternative Medicine damaged heart valves and subsequent thrombosis induction [65]. Consequent infammation and vascular changes may terminate to the functional impairment of both endothelial cells and smooth muscles of coronary arteries [69,70]. It seems that the entry of bacteria and other microbiome populations into the bloodstream is a continuous fow which inevitably leads to a surge in the expression of infammatory cytokines and chemokines. Tese factors could be drivers of CAD [71]. For example, it was shown that bacterial lipopolysaccharide (LPS) upregulates LDL levels, increasing the risk of CAD [72]. Indeed, the remnants of bacteria like DNA or membrane phospholipids provoke CAD via modulating adipose or vascular tissues [6]. However, it was reported that the development of atherosclerotic lesions is largely accelerated by live organisms rather than heat-killed ones or their LPS [73]. Reports on successful culturing of pathogens after their isolation from atheroma only existed about C. pneumoniae and E. hormaechei [74][75][76][77].
Overall, it does not clearly understand if initiation or progression of atherosclerosis is dependent on the presence of microbes. In an interesting study, the bacterial profle of atherosclerotic plaques was compared between symptomatic and asymptomatic CAD patients. Neither the amount of bacterial DNA nor microbial composition was not diferent between the two groups. It was concluded that the vulnerability of plaques may be infuenced by other factors [1]. In vitro studies demonstrated that invasive strains of P.gingivalis help foam cell formation which is a pivotal step in the evolution of atherosclerotic plaques [78]. Although the role of bacteria like Chlamydia pneumoniae has been suggested in this process [79], antibacterial therapy did not address atherosclerotic-related complications [80].
In spite of acceptable evidence on the role of microbial infection in atherosclerosis, which is derived mostly from animal models, it seems that assigning a defnitive determining role as equal as other cardiovascular risk factors for the development of atherosclerosis in humans is debated and there are numerous questions that need to be answered in this area [4].
Our study tried to cover almost all the published data on this subject. However, we should consider the possibility of unintentional mistakes to include qualifed papers among hundreds of screened papers.

Conclusion
Undoubtedly, microbes contribute substantially to human hemostasis. However, access to these elements in some sites may stimulate adverse events. It is important to elucidate the role of microbes in CAD as atherosclerosis imposes a huge health burden. Tis needs to be addressed in order to save resources for the rainy days and unprecedented diseases.

Data Availability
Te data used to support the study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.  Evidence-Based Complementary and Alternative Medicine 9