DOI: https://doi.org/10.21203/rs.3.rs-1715062/v1
This study is done to examine the microbiota in thrombi retrieved from the middle cerebral artery after mechanical thrombectomy for symptomatic carotid plaque within 6 hours of an acute ischemic stroke. To evaluate their role in atherosclerosis, thrombi were submitted to next-generation sequencing for a bacterial signature. In these study we are describing allots of bacteria found in thrombus samples, which are associated with stroke. Enrollment of 14 Acute ischemic stroke (AIS) patients was done who underwent mechanical thrombectomy in which 72% (n = 10) male and 28% (n = 4) female. The patients' median age was 16.75 ± 3.68 year old, and they were all female. During the window of acute stroke with symptomatic carotid stenosis and blockage of the ipsilateral middle cerebral artery, all were submitted to mechanical thrombectomy. After mechanical thrombectomy we extracted the DNA from thrombus sample. The microbial composition of all samples was compared using 16S rRNA gene amplicon next-generation sequencing.All of the thrombi retrieved for bacterial DNA in qPCR from the fourteen patients who had thrombectomy for ischemic stroke were positive. The 14 thrombus samples included more than 30 microorganisms. large bacterial diversity was found in thrombus in ischemic stroke patients in this study, which also indicated that microbes present in thrombus also played role in formation of plaque and Bacteria have direct mechanisms such as acidification and local inflammation of the plaque milieu with lactobacillus, biofilm dispersion leading to inflammation with pseudomonas fluorescence, or enterococci, or indirect mechanisms such as Toll 2 like signalling by the gut microbiota, could all lead to thromboembolic and cause stroke. It justifies more research into the involvement of bacteria in thrombus formation in stroke pathophysiology and prognosis.
Stroke, as the second leading cause of death and disability, has established causes of disparities and changes in trends in stroke burden across countries (1). In 2008, the yearly economic burden of informal care giving linked with stroke among the elderly in the United States was projected to be $14.2 billion (2). As previously reported, atherosclerotic plaque may have an infectious aetiology (3).The majority of previous stroke research focused on extracranial carotid atherosclerotic plaque on carotid endarterectomy patients, for example, detecting Helicobacter pylori in human carotid atherosclerotic plaques (4).
The gut microbiota appears to play a role in the aetiology and outcome of stroke, according to an increasing body of research. After an ischemic stroke, the composition of the gut flora changes. The gut microbiota, on the other hand, can affect stroke outcome and play a role in stroke causation. The gut microbiota has been associated to stroke risk factors such as hypertension, diabetes, and obesity in clinical and experimental investigations (5).
Infectious agents have been linked to persistent inflammation, which plays a role in atherosclerosis etiopathogenesis (6). Since the 1970s, researchers have been looking into the relationship between infection and atherosclerosis.
Pathogens have been linked to atherosclerosis in several studies, including (a) pathogens found in human atherosclerotic vessels (8), (b) infection linked to atherosclerosis (9) in animal studies, and (c) seroepidemiological studies showing an association between pathogen-specific antibodies and atherosclerosis (10).
Multiple pathogens, including bacteria such as Chlamydia pneumoniae, Helicobacter pylori, and periodontal pathogens, have been implicated in these associations (6).
Chlamydia pneumoniae is the pathogen whose association with atherosclerosis has received the most attention, with varying results. Chlamydia pneumoniae was the first infectious organism discovered in human atherosclerotic plaque cells, but only rarely in normal arterial cells. It is in addition one of the few agents found in plaques that have yielded viable organisms (3, 11).
A total of 14 patients with acute ischemic stroke, middle cerebral occlusion, and symptomatic carotid stenosis were admitted to Pacific Medical University & Hospital's Department of Neurology between February 2019 and March 2022.
Acute ischemic stroke with symptomatic carotid stenosis and occlusion of the middle cerebral artery, as diagnosed by neurologists, was the main criterion for inclusion. To assess ischemia lesions, all patients had a brain magnetic resonance imaging (MRI) or computed tomography (CT) scan within a 6-hour timeframe. They all had symptomatic carotid stenosis plaque, as well as a thromboembolic event that culminated in a middle cerebral artery blockage. Magnetic resonance angiograms of the neck vessel and circle of Willis vessels were conducted in addition to normal magnetic resonance imaging of the brain.
All of these carotid plaques were confirmed to be fragile or unstable plaques by morphological inspection using carotid Doppler and MRI plaque imaging. The following were among the excluding criteria: prior intravenous thrombolysis treatment; cardio embolic stroke; dissection-associated stroke; patent foramen ovale; vasculitis; history of malignancy, autoimmune disease, chronic hemodialysis kidney disease, parkinson's disease; prior intravenous thrombolysis treatment; cardio embolic stroke; hypercoagulant or genetic underlying cause of stroke; faecal thrombosis. A total of fourteen patients were enrolled in the study cohort. The National Institutes of Health Stroke Scale (NIHSS) admission rate was 16.75 ± 3.68 percent (range, 11 to 22). This study was authorized according to ethical standards by the Pacific Medical University & Hospital Regional Review Board in Rajasthan, India. The use of human participants in this study has been approved by the regional review board. All of the participants had given their informed consent in writing.
All procedures were carried out in a biplane angiography cath lab (Phillips, Allura xper FD 20 − 15). A conventional adapted seldinger approach was used to place an introducer sheath on a femoral artery, and a guiding catheter with a tip balloon up to 9F was navigated into the carotid artery proximal to the occluded location. The stent retriever was inserted using a micro catheter with a guide wire (0.021 inches) that was manoeuvred through the blocked location and over the thrombus (solitaire; Medtronic). Multiple retrieval runs were possible with Solitaire revascularization devices, with up to three retrievals in the same vessel and two retrievals per unit. The stent retrieval device was deployed by pulling the micro catheter back at the desired point after it was positioned across the visible clot. The Solitaire resheathing was done as needed, but not more than thrice. The thrombus was removed using negative aspiration using a 20 cc Syringe. The final angiography was performed to determine whether or not the flow had been restored (TICI flow). Thrombectomy was repeated until the angiographic outcome was satisfactory. The thrombus was split into a 1.5 mL eppendorf micro centrifugal tube for DNA extraction and qPCR analysis.
The DNA Extraction QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used to extract DNA from frozen thrombotic material, with minor modifications to the protocol. The technique of mechanical tissue disaggregation was developed expressly for preparing thrombotic material for DNA extraction. A NanoDrop spectrophotometer was used to determine the purity and concentration of DNA. For qPCR analysis, the minimal quality (ng/L) and purity (ratio 260/280) values were 5 and 1.5, respectively.
For the sequencing, 16S Amplicon-Seq hypervariable regions V1–V9 were amplified using primers, including the forward 16S primer 5’-ATCGCCTACCGTGAC-barcode-AGAGTTTGATCMTGGCTCAG-3’ and Reverse 16S primer 5’-ATCGCCTACCGTGAC-barcode-CGGTTACCTTGTTACGACTT-3’ with gene-specific sequences, MinION, Oxford Nanopore, and molecular barcodes. The PCR mixture for the full-length 16S rRNA gene (50 µL total volume) contained 10 ng of DNA template (10 µL), 25 µL long Amp Taq 2X master mix (NEB M0287), 1 µL 16S barcode (Barcode 01 to Barcode 04 each for a single sample separately), and 14 µL nuclease-free water as indicated in the 16S barcoding kit (SQK-RAB204). The thermal PCR profile consisted of an initial denaturation of 60 seconds at 95°C followed by 25 cycles of 20 seconds at 95°C, 30 seconds at 55°C, 2 minutes at 65°C, and a final stage of 5 minutes at 65°C. The amplicons were washed with the Beckman Coulter (AMPure XP beads) using a ratio of 0.5X. Usage of the Nanodrop spectrophotometer was evaluated for each sample quantity. The various bar-coded samples were pooled in equimolar ratio to get a final pool (100–150 ng in 10 µL) to do the sequencing library by adding 1 µL of RAP (Rapid Annealing Primer) to the bar-coded DNA. The prepared sample (11 µL DNA sample) was combined with Library Loading Beads (25.5 µL) and Fuel Mix Running Buffer (35.5 µL) and loaded onto SpotON Flow Cells Mk I (R9.4.1) (FLO-MIN106) for 12 hours using MinKNOWTM 19.06.8.
Data that support the findings of this study are available upon reasonable request from the Pacific Medical University study investigators.
We recruited 14 AIS patients who underwent mechanical thrombectomy 72% (n = 10) male and 28% (n = 4) female. The median age for the patients was 52 ± 13.6 years. All were subjected to mechanical thrombectomy during the window time of acute stroke with symptomatic carotid stenosis, with occlusion of the ipsilateral middle cerebral artery. The median delay time between the onset of ischemic stroke and hospital arrival was 6 hours.
Seven patients had a history of hypertension and 3 had diabetes mellitus. Before mechanical thrombectomy and thrombus sample collection, no patient had a history of serious infection, septicemia, or recent antibiotic treatment
Thrombus samples were collected from all participants who are going to mechanicalthrombectomy for acute stroke. Notably 100%( 14) of aspirated thrombi were positive for bacterial DNA in qPCR. More than 30 bacteria were present in 14 thrombus samples. The majority of bacteria were Staphylococcus aureus lactobacillus heliveticus, Prevotella amni, Prevotella clorans, Lactobacillus jensenni, Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus indneri, Lactobacillus, Staphylococcus, and Stenotrophomonas in all 14 thrombi. Other abundant bacteria were Staphylococcus aureus, and Bacillus cereus, Bacillus subtilis, Streptococcus pneumonia, Streptococcus pyogenes, streptococcus thermophilus and Planococcus sps. Enterococci faececium, as well as many others shown in figure. In reality, the majority of bacterial species found in abundance in (figure − 1) could be commensal or low-grade pathogens that live in close proximity to the human body. During temporary bacteremia without overt sepsis, their entry into the bloodstream from the mouth cavity, gut, or skin may cause them to become trapped in carotid plaque.
Our research found that bacterial DNA, 16 rRNA, was present in thrombi samples taken from stroke patients.
To the best of our knowledge, this is a large-cohort prospective study in which the thrombus features of acute ischemic stroke patients were fully characterized using 16S Sequencing technology, and it investigates the bacteria that are related to stroke.
There are, majority of bacteria found in the thrombus, the higher abundance of bacteria Stenotrophomonas maltophilia, Peptostreptococcus anaerobius Acinetobacter schindleri(5.77%) and Acinetobacter indicus (5.77%) while lactobacillus crispatus (20%), Lactobacillus gasseri (8%), and Lactobacillus iners (6.4%). It's possible that the majority of bacterial species in thrombus are commensal or low-grade pathogens found in the human body, which can enter the bloodstream via the mouth, gut, or skin and cause atherosclerotic plaque. Our findings reveal that next-generation sequencing can predict the relative abundance of bacteria.
Bacterial binding to platelets causes aggregation, as demonstrated by Streptococcus sanguinis, Staphylococcus epidermidis, and Chlamydia pneumoniae (12). Many bacteria, such as the gram-positive bacteria Staphylococcus aureus, can activate the blood clotting system and alter specific coagulation factors, however they do not cause the coagulation cascade to begin.
These bacteria use the coagulation system to shield themselves from being targeted by the host's immune system (13). Extracellular matrix (ECM) proteins such as fibrinogen, fibronectin, and collagen, for example, help Staphylococcus aureus form clumps (14).
Platelet aggregation development aids in the colonization of host tissues (15). Staphylococcus aureus is one of the germs that can use blood clotting to their advantage (13). Platelets, on the other hand, work along with neutrophils to create microbial traps, but this increases the risk of thrombosis (12).
Bacterial adherence to the surface of the midblock plays a key function in clot formation. Exotoxins released by Staphylococcus aureus bacteria interact with cell membranes, causing platelet activation and aggregation as well as smooth muscle contraction. Both of these causes have the potential to cause thrombosis.
As a result of the coagulase enzyme interacting with fibrinogen, plasma coagulation occurs. The most prevalent pathogen associated in venous thrombosis with osteomyelitis is Methicillin-resistant Staphylococcus aureus (MRSA). Deep vein thrombosis in children has been linked to generalized soft tissue infection, septic arthritis, osteomyelitis, and myositis (16).
MRSA infections caused higher and more severe vascular consequences than methicillin-resistant Staphylococcus aureus (MSSA) infections, especially in individuals with lung involvement. MRSA infections were also associated with higher levels of inflammation and needed longer hospital stays (17).
Many common bacteria cause infections and raise the risk of thrombotic consequences such as ischemic stroke and myocardial infarction (18, 19). Microbes also trigger an inflammatory response that involves the fibrinolytic system (20). The bacterial infection causes significant alterations in the coagulation and fibrinolysis balance (21)
Thrombosis might occur as a result of an infection that was not properly managed. However, the processes of infection-induced thrombosis, as well as the mechanisms of infection control by the host in pathogen transmission, remain poorly known.
Pathogen-induced thrombosis is related with the production of inflammatory chemicals that activate platelets, as well as damage to the endothelium, which leads to fibrin deposition and thrombus formation (22).
During the research, it was discovered that bacteria or bacterial lipopolysaccharide play a role in platelet activation and thrombus formation. Platelets can interact with gram-negative and gram-positive bacteria via direct binding via platelet membrane surface receptors and bacterial surface protein (23).
Surprisingly, changes in the distribution of bacteria patterns in thrombus from various origins were discovered. The majority of bacteria in cardioebolic stroke patients are clustered or found on the surface of red blood cells, while bacteria in LLA stroke patients are clustered or found on the surface of red blood cells (24).
However, a study found that even in the absence of atherosclerotic plaque, the presence of bacteria promotes thrombogenesis. When other bacteria congregate in a cluster, the bacillus' unique spatial localization may alter the coagulation process, which is immediately activated in blood within minutes (25).
Furthermore, some bacteria species, such as Staphylococcus aureus, release protocogulant factor, which promotes plasma coagulation to prevent lethal immune system attacks (26).
Our study showed that there are so many bacteria like bacillus and Staphylococcus aureus Pseudomonas putida, Staphylococcus epidermidis, Staphylococcus hominis, and Finegoldia magna Etc present in thrombus aspirated of stroke patients and their relative abundance that is. 3% respectively. The ability of some gram negative to build biofilms in other body locations was greatly improved (24).The Bactria in the thrombus or clot with acute ischemic stroke may play vital role in promoting blood coagulation.
Early adverse events (haemorrhagic transfusion, etc.) after mechanical thrombectomy are associated with a poor prognosis in stroke patients. The level of opportunistic pathogens such as acenatobacter and enterobacteriacy was significantly higher in patients with adverse events within 48 hours of admission than in those without.
Acinetobacter species are ubiquitous, widely distributed organisms that contribute to a variety of nosocomial illnesses (27, 28). Enterobacteriaceae enrichment in the gut is linked to an increased risk of stroke (29) and is an independent predictor of poor outcomes in stroke patients (30).
Surprisingly, when the amount of nutrients available is rapidly reduced, bacteria biofilm can be employed to respond to the resulting dispersion (31). Bacteria that form biofilms, such as Pseudomonas fluorescens and enterococcus, have been shown to raise factor VIII levels, are nonpathogenic motile, or are low-grade pathogens that increase the risk of stroke (32).
High norepinephrine enhances biofilm dispersion events, especially in reaction to stress, when a critical ingredient is liberated from its bound condition, such as free iron generation (31).
Sternotrophomonas Maltophilia is a low-virulent bacterium that forms biofilms (33). The StmPr1 gene codes for a S. maltophilia protease that can break down collagen, fibronectin, and fibrinogen protein components, potentially causing local tissue injury and bleeding (34) In periodontal disease, F. nucleatum is linked to the development of Lemierre's syndrome, a pulmonary infection accompanied by septic thrombophelebitis of the inner and outer jugular veins, arthritis, and aneurysms (35–37).
Furthermore, a larger abundance of Acinetobacter was linked to a higher 3-month mortality rate in stroke patients, according to our findings. In addition to three major clinical indications, univariate and multivariate Cox regression analysis revealed that the quantity of Acinetobacter in the thrombus was an important risk factor for 90-day death. However, the exact mechanism by which Acinetobacter has a role in poor clinical outcomes in stroke patients is unknown. Targeting the thrombus microbiota for precise regulation could help prevent thrombosis, reduce the risk of stroke, and improve the clinical outcome of stroke patients.
The study showed evidence for significant of diversity of bacteria in thrombus in ischemic stroke patients. Our study indicated that microbes present in thrombus which is formation of plaque as well as associated with stroke .It is among the first fully characterize the thrombus microbial feature observe which is closely associated with pathogens and risk of death receiving mechanical thrombectomy. It is further investigation on the role of bacteria in thrombus in the pathogenesis and prognosis of stroke
Ethical Approval and Consent to participate
Not applicable.
Consent for publication
Not applicable.
Availability of data and materials
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Funding
The study has been funded by the Department of Science and Technology Govt. of Rajasthan India.
Authors' contributions
LBY, and AV, ST designed and wrote the manuscript. ST, LBY, and MV, revised the article. All authors have read and approved the manuscript. LBY, AV,, ST, and, contributed equally.
Acknowledgement
The Pacific Medical University and Hospital provided the authors with facilities and resources during the research. The writers gratefully acknowledge financial support from the Rajasthan Government's Department of Science and Technology.
Table-1
|
Patient characteristics |
PT1 |
PT 2 |
PT 3 |
PT 4 |
PT 5 |
PT6 |
PT 7 |
PT 8 |
PT 9 |
PT10 |
PT11 |
PT12 |
PT13 |
PT14 |
|
Age /Gender |
46 M |
53M |
70M |
33M |
50F |
30F |
55F |
40M |
51M |
43F |
51M |
49M |
43M |
62M |
|
IV Thrombolysis |
YES |
YES |
YES |
YES |
YES |
YES |
NO |
NO |
YES |
YES |
YES |
NO |
YES |
NO |
|
NIHSS/ admission |
15 |
13 |
17 |
12 |
10 |
19 |
2 |
11 |
14 |
13 |
18 |
21 |
22 |
19 |
|
MRS/ admission |
4 |
2 |
4 |
3 |
2 |
4 |
3 |
3 |
2 |
4 |
4 |
5 |
6 |
9 |
|
Duration of samples from onset /hour |
6 |
4 |
3 |
4 |
3 |
5 |
2 |
4 |
5.5 |
3.6 |
2 |
5 |
6 |
3 |
|
Diabetes |
NO |
NO |
YES |
NO |
YES |
NO |
NO |
YES |
NO |
NO |
NO |
NO |
NO |
NO |
|
Hypertension |
NO |
YES |
YES |
NO |
YES |
NO |
YES |
NO |
NO |
NO |
YES |
NO |
YES |
YES |