P-110 - METAGENOMICS EXPLORING THE BACTERIOME IN CAROTID STENOSIS: A PILOT STUDY

TOPIC:
Other
AUTHORS:
Emma P. (Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Research Group. Medical Research Institute Hospital La Fe ~ Valencia ~ Spain) , Alessandra F. (Universitat de València-I2sysbio-FISABIO-Public Health Mixed Unit in Genomics and Health ~ Valencia ~ Spain) , Nuria J. (CIBERESP (Instituto de Salud Carlos III) and Universitat de València-I2sysbio-FISABIO-Public Health Mixed Unit in Genomics and Health ~ Valencia ~ Spain) , Miguel C. (Universitat de València-I2sysbio-FISABIO-Public Health Mixed Unit in Genomics and Health ~ Valencia ~ Spain) , Andrés M. (CIBERESP (Instituto de Salud Carlos III) and Universitat de València-I2sysbio-FISABIO-Public Health Mixed Unit in Genomics and Health ~ Valencia ~ Spain) , Alejandro M. (CIBERESP (Instituto de Salud Carlos III) and Universitat de València-I2sysbio-FISABIO-Public Health Mixed Unit in Genomics and Health ~ Valencia ~ Spain) , Francisco Xavier L. (CIBERESP (Instituto de Salud Carlos III) and Universitat de València-I2sysbio-FISABIO-Public Health Mixed Unit in Genomics and Health, and Department of Microbiology and Ecology, Medical School, Universitat de València ~ Valencia ~ Spain) , Manuel M. (Angiology and Vascular Surgery Service. University and Polytechnic Hospital La Fe ~ Valencia ~ Spain)
Introduction:
Atherosclerosis is a complex disorder with a high inflammatory contribution. The association between bacterial infection and atherosclerosis has been suggested for decades and even some studies based on standard technologies have detected some pathogens in carotid specimens (1). Nevertheless, current techniques based on genetic amplification and high-throughput sequencing (metagenomics) allows for simultaneously studying all the bacterial burden from a single sample. Moreover, the study of the plaque microbiome could help to better understand mechanisms related with plaque formation and rupture. Consequently, our main objective was to investigate the applicability of metagenomics to explore bacterial DNA present in atheroma plaque and serum from patients with carotid stenosis.
Methods:
Tissue and serum samples were obtained from patients with carotid stenosis that underwent carotid endarterectomy. Symptomatic (N=14) and asymptomatic (N=27) patients, sex and age matched were recruited. Symptomatology was considered when stroke, transient ischaemic attack or amaurosis fugax occurred within <21 days before surgery. Plaque specimens were snap frozen during surgery and stored at N2 until use. A fragment of 150 mg was excised under sterile conditions. Blood samples were extracted during surgery, clot was formed after 1h incubation at room temperature, and serum was obtained by centrifuging at 1,800 x g at 4ºC. Nucleic acids were extracted from plaques and serum samples after enzymatic digestion and homogenization using the Nuclisens eMAG automated platform with a previous incubation with collagenase in case of plaque samples. Bacterial ribosomal DNA (V3-V4-16sr DNA) was amplified and sequenced by 16s metagenomics (Illumina MiSeq v3). The bioinformatic and taxonomic analysis of 16s rDNA was performed with Dada2 and R-Statistics. A ratio >10 sequences from samples to negative controls was required to discard false positives caused by contamination.
Results:
Nucleic acids from bacteria were hardly detected in plaque and serum samples. Nevertheless, the following bacterial genus were found in plaque (N asymptomatic vs symptomatic): Staphylococcus (4 vs 5), Uruburella (10 vs 4), Sphingomonas (1 vs 0), Strenothropomonas (2 vs 0), Acinetobacter (0 vs 1) and Alishewamella (0 vs 1). In serum samples the bacterial genus detected (N asymptomatic vs symptomatic) were: Uruburuella (6 vs 2), Escherichia/Shigella (6 vs 2), Acinetobacter (2 vs 0), Anaerobacillus (7 vs 0), Bacillus (3 vs 3), Bukholderia (5 vs 5), Parabacteroides (2 vs 0), Paracoccus (1 vs 0), Pseudomonas (6 vs 1), Staphylococccus (1 vs 1) and Streptomyces (13 vs 9).
Conclusion:
These preliminary results demonstrate the feasibility of metagenomics to detect presence and relative abundance of bacterial species in atheroma plaques and serum from patients with carotid stenosis. Larger studies will allow us to explore the relationship between pathogenic burden and plaque stability, and therefore with the risk of ischaemic events.
References:
Campbell LA et al. Infection and Atherosclerosis Development. Arch Med Res. 2015;46(5):339-50. ISCIII-FEDER (PI20/01171)