Application of high-throughput sequencing to the study of the main bacterial populations in carotid stenosis.

Abstract

Introduction: The potential involvement of pathogens in the development of atherosclerosis has been studied for decades. Some previous studies have successfully identified the presence of pathogens in the atheromatous plaque. However, many of these determinations are aimed at detecting the presence of a particular species. The use of omics technologies allows for the analysis of the complete microbial profile of a given sample. In the specific case of atheromatous plaque, the study of the bacterial load composition would help to clarify the possible relationship between infection and atherosclerosis and identify whether there is a bacterial profile associated with unstable plaques and, therefore, with the consequent risk of ischemic events.

Methodology: We analyzed cross-sectional fragments of carotid atheromatous plaque (N=57) and serum (N=54) from patients with recent neurological symptoms and asymptomatic patients (control group). Nucleic acids were extracted from the samples by enzymatic digestion and homogenization, with additional treatment with type I collagenase in the case of plaques. Bacterial ribosomal RNA (16S-rRNA gene) was amplified and subjected to massive sequencing using the Illumina® Miseq platform. The bioinformatic analysis, to identify the taxonomic composition, and biostatistical analysis, to determine the significant taxa, of the 16S-rRNA was performed in the R environment. As contamination control, bacterial species ratios ≥10 with respect to negative controls were considered significant.

Results: The presence of bacterial 16S-rRNA was very low in both types of samples. The bacterial composition in terms of α diversity and β diversity differed between plaque and serum; however, we did not observe significant differences between samples from symptomatic and asymptomatic patients. The most abundant phylum and genus in plaque were Firmicutes and Staphylococcus, respectively. For Staphylococcus, we found 100% similarity homology of the 16S-rRNA with 3 species (S. epidermidis, S. caprae, and S. capitis). In serum, the most abundant phyla were Firmicutes and Proteobacteria, with Streptococcus being the dominant genus, for which we found 100% homology of the 16S-rRNA with 20 species of oral streptococci.

Conclusions: We have successfully applied massive sequencing techniques to determine the presence and relative abundance of bacterial species in atheromatous plaques and serum of patients undergoing carotid endarterectomy. We have not observed significant differences between symptomatic and asymptomatic patients regarding the main genera, so we cannot establish a direct connection between bacterial composition and atheromatous plaque vulnerability. However, a possible association between atherosclerosis and the presence of staphylococci and streptococci in plaque and serum, respectively, cannot be ruled out.