Iranian Journal of Medical Microbiology

 
Table 1. List of Primers for DGGE Assay

I, inosine.
 

DGGE Assay

PCR products were loaded on the polyacrylamide gel and DGGE was performed for 17h at 60V and 60˚C, according to previously published papers [16, 18].

Sequencing

The eligible bands were cut with a scalpel and PCR product was extracted using a gel extraction kit. The sequencing process was carried out by Pishgam Company (Iran).

Statistical analysis

After data collection, the data were analyzed using SPSS version 19 (Chicago, IL, USA). The Chi-square test was used to determine the correlation between variables, and the t-test was used to determine logistic regression and odds ratio. The significance level of statistical tests was set sat less than 0.05.

It has been shown that oral microbiota can enter  the bloodstream and localize in atherosclerotic lesions (17, 18). Several studies confirm the relationship between metabolic parameters and increased risk of atherosclerosis (19, 20). In our study,  we detected bacterial DNA in atherosclerotic plaques in 64% of the samples, possibly due to the method used for bacteria detection (21, 22). We developed DGGE assay to the investigate relationship between metabolic parameters and oral microbiota composition in patients with atherosclerosis. DGGE assays have been used to identify multiple different single-base substitutions in a variety of sequences (23, 24).
Out of 139 saliva samples collected, 89 people (64%) had arteriosclerosis and 50 people (36%) were healthy. The mean age of patients with atherosclerosis was 62.71 years and healthy individuals was 57.88 years. Also, 46.6% (90) of the participants were male and 25.4% were female. The BMI of 47.1% of atherosclerosis patients and 54% of healthy people in this study was 20-25.  These results disagree with other studies, which could be due to the low sample size, different geographical areas, and lifestyles.  There are considerable differences between the mean levels of total cholesterol, LDL, FBS, and BUN in the two groups of control and atherosclerosis patients. However, there was no significant relationship in the mean levels of Triglyceride and HDL between the two groups, which is consistent with other studies by Kron et al., Xian et al. and Abbate et al. (22, 25, 26). 
DGGE assay was used to study the profile of bacteria in the oral cavity, which showed that The LDL levels in the two groups with and without S. salivarius was significantly different (P-value <0.05). This means that people who have the S. salivarius in their mouth have a lower and normal average LDL, whereas people who do not have this bacterium in their mouth have a higher LDL. This is consistent with Calandrini et al. study (27).  There was also no significant difference in HDL levels between the two groups, which disagreed with another study, possibly due to the small sample size (139 samples) compared to other studies. Moreover, significant differences exist between the FBS in the two groups with Actinomyces and B. culaenoe (P-value <0.05). This means that people who have Actinomyces and B. culaenoe in their mouth have a lower and normal average FBS, whereas people who do not have these bacteria in their mouth have a higher average FBS. Similar results were reported by Grau et al (28). Our result shown that cholesterol levels in the two groups with and without Actinomyces and N. perflava are significantly different. This means that people who have Actinomyces, and N. perflava in their mouths have average to low cholesterol, whereas other people have higher cholesterol averages. These results were agreement to Niemi et al., Leishman et al., and Calandrini et al. (27, 29, 30) Additionally, similar to a study by Kato-Kogoe N's et al (31), there was no significant relationship between the mean triglycerides levels of the two groups. On the other hand, BUN levels in the two groups with and without N. perflava was significantly different (P-value <0.05). This means that people who have N. perflava in their mouth have a lower and normal average BUN, whereas people who do not have this bacterium in their mouth have a higher average BUN, which is consistent with the findings of Bouzid et al. study (32). According to our result and several studies, these bacteria or their products probably caused the decrease in FBS, cholesterol, BUN, etc (33), which is due to a favorable condition for growth of oral cavity bacteria such as Veillonella atypica, Porphyromonas gingivalis, Prevotella intermedia, and Streptococcus faecalis (34, 35). Many non-culturable organisms which are only identifiable by molecular assay, reside in the oral cavity (36).
One of the limitations of this study is the low sample size. The DGGE assay can be used as an effectual screening assay to investigate the relationship between metabolic parameters and oral microbiota composition in patients with atherosclerosis.

 

Our data shown DGGE assay is a good way to diagnose and compare the oral microbiota of people with atherosclerosis and healthy. Thus, the oral microbiota should be further examined to determine its potential as a biomarker for atherosclerosis.

 

The authors would like to thank the staff of the Department of Microbiology, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran.

 

None.

 

Conflicts of Interest

The authors declare no conflict of interest.