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The Presence of Porphyromonas Gingivalis, Chlamydia Pneumonia

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The Presence of Porphyromonas Gingivalis, Chlamydia Pneumonia Journal of Research in Medical and Dental Sciences 2018, Volume 6, Issue 3, Page No: 1-6 Copyright CC BY-NC-ND 4.0 Available Online at: www.jrmds.in eISSN No. 2347-2367: pISSN No. 2347-2545 The Presence of Porphyromonas Gingivalis , Chlamydia Pneumonia , Helicobacter Pylori , Mycoplasma Pneumonia and Enterobacter Hormaechei DNA in the Atherosclerotic Plaques Saeed Shirgir Abibiglou 1, 2, Hossein Bannazadeh Baghi 1, 2, Mohammad Yousef Memar 2, 3, Nasser Safaei 4, Rezayat Parvizi 4, Mansur Banani 5, Naser Alizadeh 1,2 , Reza Ghotaslou 1, 2* 1Immunology Research Centre, Tabriz University of Medical Sciences, Tabriz, Iran 2Department of Microbiology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran 3Students’ Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran 4Department of Cardiothoracic Surgery, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran 5Department of Parasite and Fungal diseases, Razi Institute, Iran DOI: 10.5455/jrmds.2018631 ABSTRACT Cardiovascular disease is the most common illness in the developed and developing countries. Atherosclerosis is a cardiovascular inflammatory disease, which causes tissue destruction and fibrosis in the long run. On the other hand, atherosclerosis is a very common disease and is a multifactorial process. A potential role of some infectious agents has been suggested in the pathogenesis of atherosclerosis. This study aimed to determine the presence of Porphyromonas gingivalis, Chlamydia pneumonia, Helicobacter pylori, Mycoplasma pneumonia and Enterobacter hormaechei DNA in coronary artery atherosclerotic plaques by PCR analysis and the study of association between the presence of bacterial DNA and atherosclerosis, clinical, and demographic features of patients. Twenty-eight patients with atherosclerotic diseases who had undergone coronary artery bypass grafting surgery were included. Cinna Pure DNA Kit was used for extraction of total bacterial genomes. The presence of bacterial DNA in endarterectomy specimens were detected by the PCR using 16S rRNA and conserved specific primers. The significant statistic association between presence of different bacterial DNA and clinical and laboratory features of included patients were evaluated by χ2 and Fisher’s exact tests. C. pneumoniae and H. pylori DNA was detected in 4 out of 28 (14.3%) and 1 of 28 (3.6%) atherosclerotic plaques, respectively. DNA of Mycoplasma pneumonia, Enterobacter hormaechei and Porphyromonas gingivalis were not detected. There was no considerable difference between the presence of bacterial DNA and clinical and demographic features of patients. The higher rate of Chlamydia pneumoniae and Helicobacter pylori DNA in atherosclerotic damages suggest that these pathogens may play an important role in the pathogenesis of atherosclerosis progression. Key words: Atherosclerosis, Chlamydia pneumonia , Helicobacter pylori HOW TO CITE THIS ARTICLE : Saeed Shirgir Abibiglou, Hossein Bannazadeh Baghi, Mohammad Yousef Memar, Nasser Safaei, Rezayat Parvizi, Mansur Banani, Naser Alizadeh, Reza Ghotaslou *, The presence of Porphyromonas gingivalis, Chlamydia pneumonia, Helicobacter pylori, Mycoplasma pneumonia and Enterobacter hormaechei DNA in the atherosclerotic plaques, J Res Med Dent Sci, 2018, 6 (3):1-6, DOI: 10.5455/ jrmds.20186631 Corresponding author : Reza Ghotaslou disease, which causes tissue destruction and e-mail : [email protected] fibrosis in the long run [2, 3]. Atherosclerosis is a Received: 02/02/2018 Accepted: 19/02/2018 very common disease and is a multifactorial process. It is considered to be one of the chief INTRODUCTION causes of morbidity and mortality [4-6]. Several studies have reported evidence indicating that Cardiovascular disease is the most common illness infection is a risk factor for atherosclerosis and in the developed and developing countries [1]. plays an important role in chronic inflammatory Atherosclerosis is a cardiovascular inflammatory progressions either via direct or indirect Journal of Research in Med ical and Dental Science | Vol. 6 | Issue 3 | Ma y 2018 1 Reza Ghotaslou et al J Res Med Dent Sci, 2018, 6 (3):1-6 _____________________________________________________________________________________ mechanisms [7, 8]. The association of bacterial DNA extraction and viral pathogens with atherosclerosis has been After homogenization with a manual homogenizer, illustrated by sero-epidemiological assessments samples were incubated for 2 h in a that indicate a higher risk of atherosclerosis with SDS/proteinase K solution at 37°C. The total DNA increased levels of antibodies against specific was extracted by Cinna Pure DNA Kit. pathogens as well as the detection of infectious agents in atherosclerotic lesions. However, the Detection of bacterial DNA burden of evidence for a contributing role of The universal bacterial 16sRNA was used for the infectious agents lies in establishing biological detection of bacterial DNA. Conserved specific plausibility that the pathogen contributes to primers were used for the detection of atherosclerotic progression and therefore, that Porphyromonas gingivalis (P. gingivalis ), therapeutic strategy or protective approaches Chlamydia pneumonia (C. pneumonia ), influence disease development [2]. The detailed Helicobacter pylori (H. pylori), Mycoplasma mechanism of atherosclerosis, which is the main pneumonia (M. pneumonia) and Enterobacter cause of this disease, is the progression of chronic hormaechei (E. hormaechei) . PCR conditions and inflammation has been established as a common primer sequences were shown in table-1. Positive theory, but its association with the particular and negative controls, obtained by the addition of infectious agent (which can induce such chronic genomic DNA of bacteria or sterile distilled water, inflammations) has not been known until now[1]. respectively, were used in every set of reactions. Several individual bacterial pathogens have been The PCR products were submitted to detected more commonly in atherosclerotic electrophoresis in 1/5% agarose gel.[9]The PCR legions by means of electron microscopy, products were sequenced for confirmation of the immunocytochemistry, and polymerase chain presence of bacterial DNA. reaction (PCR) that suggest their role as agents of the endothelial injury result in atherogenesis [7]. Statistical analysis In the present study, the relationship between the The relationship in the presence of different kinds presence of several bacterial DNA in of bacterial DNA and clinical and laboratory atherosclerosis plaque and cardiovascular risk characters were evaluated by using χ2 and factors in Iranian adult patients who had Fisher’s exact tests by the SPSS software undergone surgery were investigated. (Washington, the USA), version 19. A P-value of ≤ 0.05 was considered statistically significant. MATERIALS AND METHODS RESULTS Patients and sample collection The subjects, included in this study, were patients In the present study, 28 specimens of atheroma attending Shahid Madani Medical Research and plaques were collected from 22 male and 6 female Training Hospital, Tabriz who had undergone patients with a mean age of 55±10.41 years and surgery and the atherom was taken. Information were examined for the presence of bacterial DNA. about epidemiological data and other risk factors The total amount of 80 to 230 ng of genomic DNA were recorded and 28 subjects with was extracted from each specimen. By using the atherosclerosis were included. We used a piece of universal bacterial 16sRNA primers it was found non-atherosclerotic artery (grafts) as a control of that 39% of inspected specimens were positive for the study. This study was approved by the Ethics bacterial DNA presences. In the present study, H. Commission of Tabriz University of Medical pylori and C. pneumoniae DNA was detected in Sciences. Atheromatous plaque samples and non- specimens one (3.6%) and 4 (14.3%) through the atherosclerotic artery were dissected in the use of conserved specific primers for the PCR operating room and placed in a 5 ml TE buffer (50 (figure 1 and 2). All of them were positive for mM Tris/ HCl, pH 7.6; 1 mM EDTA, pH 8.0) under 16sRNA. DNA of Mycoplasma pneumonia, sterile conditions. Samples were immediately Enterobacter hormaechei and Porphyromonas frozen at -20°C and were stored at -80°C until gingivalis was not detected by PCR using specific processing and were manipulated by the same conserved primers. In our study, there was no investigator . significant difference between the presence of bacterial DNA and clinical and demographic features of patients (Table-2). Interestingly, hyper-triglyceridemia (P-value 0.42) was found in Journal of Research in Med ical and Dental Science | Vol. 6 | Issue 3 | Ma y 2018 2 Reza Ghotaslou et al J Res Med Dent Sci, 2018, 6 (3):1-6 _____________________________________________________________________________________ all (100%) infected and non-infected patients. evidence of a significant role in the pathogenesis Hypercholesterolemia (P-value 0.2) and of atherosclerosis. [2] hyperglycemia (P-value 1) was observed in 5 (45.5%) and 8 (72.7%) of infected patients and 11 (64.7%) and 11 (64.7%) in non-infected patients, respectively. Table 1: PCR assays for detection of bacterial DNA PCR PCR target Primers sequences Ref condition 94ºc -45s C. F: 5'- GTTGTTCATGAAGGCCTACT-3' 50ºc-30s [11] pneumoniae R: 5'-TGCATAACCTACGGTGTGTT-3' 72ºc-30s (40 cycles) 94ºc-1 min F: AAGCTTTTAGGGGTGTTAGGGGTT- 56ºc-1 min [11] H. pylori 3' 72ºc-1 min R:
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