- Department of Neurological Surgery, University of Washington, Seattle, Washington, United States.
- Genomic Medicine Group, J. Craig Venter Institute, La Jolla, California, United States.
Melanie Walker, Department of Neurological Surgery, University of Washington, Seattle, Washington, United States.
DOI:10.25259/SNI_104_2022Copyright: © 2022 Surgical Neurology International This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.
How to cite this article: Melanie Walker1, Carla Uranga2, Samuel HS Levy1, Cory Kelly1, Anna Edlund2. Thrombus-associated microbiota in acute ischemic stroke patients. 10-Jun-2022;13:247
How to cite this URL: Melanie Walker1, Carla Uranga2, Samuel HS Levy1, Cory Kelly1, Anna Edlund2. Thrombus-associated microbiota in acute ischemic stroke patients. 10-Jun-2022;13:247. Available from: https://surgicalneurologyint.com/surgicalint-articles/11643/
Background: Despite a reduction in stroke incidence and age-standardized death rates, stroke remains a leading cause of death and disability worldwide. Significant interest in recent years has focused on the microbiota-host interaction because accumulating evidence has revealed myriad ways in which bacteria may contribute to risk of stroke and adverse outcomes after stroke. The emergence of endovascular thrombectomy as a treatment provides a unique opportunity to utilize thrombus retrieved from cerebral arteries to fill knowledge gaps about the influence of bacteria on stroke pathophysiology. While bacterial signatures have been confirmed in cerebral thrombi, the exact nature of the pathogenesis has not been established.
Methods: Thrombi were obtained from a cohort of adult ischemic stroke patients during standard of care thrombectomy. After DNA extraction and quantification, thrombi underwent 16S rRNA amplicon-based metagenomic sequencing, followed by bioinformatics processing. Taxonomic identification of bacterial colonies isolated on Agar plates from plated suspension was performed using DNA extraction and full length 16S Sanger sequencing.
Results: A broad diversity of bacterial signatures was identified in specimens, primarily of cariogenic origin.
Conclusion: In this small study, we demonstrate proof of concept and technical feasibility for amplicon-based metagenomic sequencing of arterial thrombi and briefly discuss preliminary findings, challenges, and near-term translational opportunities for thrombus genomics.
Keywords: Metagenomics, Microbiota, Stroke, Thrombectomy
Accumulating evidence suggests that the human microbiota may influence the development or outcomes related to acute ischemic stroke (AIS). Cerebral thrombi represent a new source of biological information and may provide insight into the vascular microenvironment. No standards or guidelines exist for next generation sequencing (NGS) of thrombus-associated microbiota. To demonstrate proof of concept and technical feasibility in this setting, cerebral thrombi were subjected to amplicon-based bacterial 16S rRNA gene sequencing.
Subjects, procedure, and specimens
Thrombi were obtained from cerebral arteries of subjects over age 18 with AIS during standard-of-care endovascular thrombectomy (EVT) at a Comprehensive Stroke Center between January 2020 and October 2020. Analysis was limited to subjects whose thrombi were obtained intact, with full reperfusion (mTICI = 3).[
During the thrombectomy, a combined technique was used. In addition to stent retriever, aspiration was applied through an intermediate catheter as well as a large-bore guide catheter positioned in the cervical carotid to help ensure first pass reperfusion. Because the thrombus is frequently adherent to the devices, it must be washed with sterile saline and gently manipulated for transfer into the sterile collection receptacle. The thrombus was rinsed with sterile water, placed into a pre-prepared sterile collection container containing phosphate buffered saline (PBS), and stored at −80°C until processing.
DNA extraction and quantification
After homogenization, DNA extraction was performed using the Ultra-Deep Microbiome Prep protocol (Molzym, Portland, OR). DNA was quantified using Qubit® dsDNA high-sensitivity and broad-range fluorometric assays (Thermo Fisher Scientific, Waltham, MA). DNA quality was assessed using Agilent 2100 Bioanalyzer technology (Agilent, Santa Clara, CA).
Analysis of thrombus microbiota and quality control
Multiplex 16S rRNA gene (16S) fragment amplification (2 × 300bp paired end) using the Swift Amplicon® panel (SWIFT Biosciences, Ann Arbor, MI) that targets all nine variable regions (V1-V9) of bacterial and archaeal 16S was performed, followed by Swift 2S® Turbo DNA library preparation (SWIFT Biosciences) and deep sequencing through MiSeq® platform (Illumina Inc. La Jolla, CA). Negative controls (water) were included in all sample preparation and sequencing experiments. The R-based DADA2 open-source software package next-generation microbiome bioinformatics algorithm[
The mean age of subjects was 62, with a standard deviation of 18.8 years. Although all were male, four unique racial/ ethnicities were represented.[
Bacterial DNA identified in cerebral thrombi
Deep sequencing of forward and reverse sequence reads representing all nine variable regions of the bacterial 16S amplified from DNA revealed the presence of bacterial signatures. The main bacterial groups associated with specimens in this cohort belonged to the Acetobacter, Streptococcus, and Lactobacillus genera [
Biplot analysis of bacterial taxa based on 16S fragment sequencing (a and b) reveals the presence of a diverse bacterial community within cerebral thrombi. Signatures of oral pathogens, specifically cariogenic Staphylococcus, Streptococcus, and Lactobacillus were revealed in thrombi (c and d).
Validation of prior reports
Analysis of the thrombus-associated microbiota in this cohort supports findings from several previous studies, which report oral bacterial signatures in cerebral,[
Several technical challenges were present, including low biomass specimens, insufficient specimen controls, and sample preservation conditions. Low-biomass samples with high non-microbial (host) nucleic acids may yield small quantities of bacterial DNA that may be insufficient for library construction.[
Analysis of the thrombus microbiota from cerebral thrombi is technically feasible and demonstrated the presence of multiple taxonomic groups. The confirmation of bacterial signatures representing the human microbiota, specifically the oral microbiota,[
Institutional Review Board (IRB) permission obtained for the study.
There are no conflicts of interest.
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