Overexpansion of Proteobacteria in the Thrombus of Stroke Patients Treated with Mechanical Thrombectomy and the Risk for Death
Authors: Liao Yu , Zeng Xiu-li , Xie Xiao-mei , Liang Dan , Qiao Hong-yu , Wang Wen-ce , Guan Min , Huang Sheng-ming , Jing Zhen , Leng Xin-yi , Huang Li'an 
Published: May 2021

Abstract
Background: Extensive analysis has focused on the brain-gut axis interaction between stroke and the gut, but the most direct cause of stroke involves thrombi that block blood flow. The role of bacteria in thrombi on stroke onset has rarely been studied. Thus, it is of interest to fully characterize the microbial features of the thrombus and explore the underlying mechanism.Materials and methods: Clot samples were collected from 104 acute ischemic stroke (AIS) patients who underwent clot retrieval with mechanical thrombectomy. After inclusion, oral, fecal, and isolated plasma samples of the same patients were synchronously gathered within 12 hours of admission. The microbial composition of all samples was compared using 16S rRNA gene amplicon next-generation sequencing. Fluorescent in situ hybridization (FISH) was selected to detect the bacteria in the thrombus with the bacterial probe EUB338. The markers of inflammatory cytokines and intestinal barrier integrity were measured using ELISAs. We performed a comprehensive analysis of the characteristics of the thrombus bacterial community in stroke patients and calculated the correlation between specific bacterial genera and clinical outcomes.Results: Of the 104 AIS patients, the presence of bacterial DNA in the thrombus was confirmed by qPCR analysis of the 16S rRNA gene and FISH experiments. Operational taxonomic units (OTUs) from 14 different phyla were identified, with the majority of the OTUs belonging to Proteobacteria (73.3%); these were mainly predicted by BugBase to be potential opportunistic pathogens. Bray-Curtis distance analysis revealed that the microbiota in the thrombus were significantly different from the oral, fecal and plasma microbiota (all P<0.001, Adonis test). A proportion of the clot microbiome was similar to that of plasma, and based on the FEAST algorithm, the bacteria in the thrombus were mainly derived from plasma (46.69%). Higher abundances of Acinetobacter and Enterobacteriaceae were associated with a risk of adverse events within 48 hours of admission, and a higher abundance of Acinetobacter was also associated with a risk of death by the 3rd month. Univariate and multivariate Cox regression analysis showed that, in addition to important clinical indicators (preoperative National Institutes of Health Stroke Scale (NIHSS) score, atrial fibrillation and history of stroke), the relative abundance of Acinetobacter in the thrombus was an important risk factor for 90-day mortality (HR 1.957, 95% CI 1.198-3.196, P=0.007; adjusted HR 2.664, 95% CI 1.384-5.129, P=0.003). Higher concentrations of IL 1β and IL 6 were associated with a higher risk of adverse events and death.Conclusion: The study showed evidence for significant diversity of bacteria in the thrombus in ischemic stroke patients and visually confirmed their adherence in FISH. It is among the first to fully characterize the clot microbial feature as overexpansion of the phylum Proteobacteria and observe a close correlation between conditional pathogens and risk of death in patients receiving mechanical thrombectomy. It warrants further investigations on the role of bacteria in the thrombus in the pathogenesis and prognosis of stroke.