Clots retrieved by mechanical thrombectomy from acute ischemic stroke patients show no evidence of bacteria

Abstract

Background

Bacteria and bacterial components have been associated with the activation of coagulation factors and initiating the blood clot formation. The aim of this study was to investigate whether bacterial populations are present in clots retrieved from patients that have suffered a large vessel occlusion acute ischemic stroke (AIS).

Materials and methods

Clot samples were collected from 20 AIS patients who underwent clot retrieval with mechanical thrombectomy. Patient clinical demographic details were noted. Expression of bacterial 16S rDNA was analyzed by standard and real-time polymerase chain reaction (PCR). Gram staining was performed to identify Gram-positive and Gram-negative bacteria.

Results

Both the real-time and standard PCR demonstrated no expression of 16S rDNA in any of the 20 clots samples from AIS patients. Gram staining results showed no expression of Gram-positive or Gram-negative bacteria present in the clot samples.

Conclusion

Our current study found no bacteria populations in the clots of AIS patients.

Introduction

Acute ischemic stroke (AIS) accounts for >80% of all strokes.¹ The presence of bacterial, viral or fungal infections have been noted in the vascular wall and atherosclerotic plaque in peripheral arteries.^2,3 It is known that bacterial infections result in immunological reactions which initiate blood clotting.^2,4 Several bacteria and bacterial components can activate coagulation factors^5–8 and initiate the coagulation cascade that precedes blood clot formation, and clusters of bacteria have the capability to directly initiate coagulation during infection within minutes. Furthermore, bacteria can pass the coagulation threshold and can propagate the clot formation.⁴

The pathogenesis of ischemic stroke may result from the infectious and aberrant activation of the coagulation process and endothelial dysfunction.^9–11 Chronic infection such as sepsis or septicemia in the context of stroke risk is well known.^12,13 Septicemia initiates the systemic inflammatory reaction and dysregulates the coagulation process, resulting in structural changes in the cerebral vasculature, thereby increasing the risk of developing both ischemic and hemorrhagic strokes.¹² Additionally, the presence of bacteria such as Chlamydia pneumoniae in atherosclerotic plaques, abdominal aortic aneurysms and cardiovascular diseases is well established.^2,14,15 Based on this prior evidence, we hypothesized that bacteria would be found within clot samples based on the fact that they are often present in atherosclerotic plaques. The aim of the current study was to investigate whether bacteria are present in clots retrieved from patients who have recently suffered an AIS.

Materials and methods

Patient cohort and clot sample collection

Twenty AIS patients met the inclusion criteria and were included in the study. Inclusion criteria include: (a) adult patients (>18 years); (b) having undergone mechanical thrombectomy with retrieval of clot material who (c) have a clot available for bacterial and histopathological analysis; and (d) did not present with stroke in the context of septicemia or endocarditis. AIS patients who did not have a clot available for analysis were excluded from the study. Information regarding demographics, use of intravenous or intra-arterial recombinant tissue plasminogen activator (rtPA), final angiographic outcome, number of passes needed, and list of devices used were noted. The institutional review board approved the study (ID No. 16-001131-7/5/2016 Stroke Thromboembolism Registry of Imaging and Pathology).

On retrieval, the AIS patients’ clot samples were immediately placed in saline. On the same day, the sample was cut longitudinally into two parts; one part of the sample was immediately used for isolation of DNA and the other part was fixed in formalin for histology. All of the clot collection and preparation processes were carried out under sterile conditions.

Isolation of DNA and polymerase chain reaction amplification

DNA was isolated from the clots using a PureLink Genomic DNA Mini Kit (Invitrogen, CA) as per the manufacturer’s instructions. The DNA concentration was determined using a NanoDrop 2000c (Thermo Scientific, WI). Polymerase chain reaction (PCR) amplification of the bacterial-specific 16S rDNA was performed using universal bacterial primer pair 5′-CCAGACTCCTACGGGAGGCAGC-3′ and 5′-CTTGTGCGGGCCC-3′ at 95℃ for 5 min, 94℃ for 1 min, 65℃ for 1 min and 72℃ for 1 min for 32 cycles. These primers cover the hypervariable region of V3-V5 of the 16S rDNA gene.¹⁵ Escherichia coli genomic DNA (gDNA) was used as a positive control and sterile water was used as the negative control. The amplicons were run in 0.8% agarose gel by electrophoresis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Real-time quantitative PCR amplification

16S rDNA gene quantitative PCRs (qPCRs) were performed with SYBR GreenER qPCR Supermix (Invitrogen, CA) at annealing temperature 65℃ for 1 min for 32 cycles (Bio-Rad, CFX Connect, Real-Time system). Positive and negative controls were used for the quantification. The SYBR Green qPCR product was run in 1.5% agarose gel by electrophoresis. GAPDH was used as a loading control at an annealing temperature of 58℃ for 39 cycles. To confirm further the presence or absence of bacterial communities, gDNA samples were sent to the Microbiome Department, University of Minnesota, for 16S material amplification and sequencing.

Gram bacterial staining

The formalin-fixed clot samples were processed overnight in a tissue processor (Leica ASP300s) and paraffin embedded. The formalin-fixed and paraffin-embedded clot samples were cut into 5 -µm slices and mounted on glass slides. Prior to staining, the slides were incubated at 60℃ for 30 min to melt the paraffin and then rehydrated through changes of xylene, alcohol (100%, 95% and 80%) and water for 5 min each. The slides were then stained with the Gram staining kit according to the manufacturer’s instructions (Thermo Scientific Richard–Allan Scientific Chromaview Advanced testing Gram stain-film kit). Staphylococcus aureus and E. coli were used as positive control. The clot sections were examined and imaged (Imaging, MicroPublisher 5.0 RTV).

Statistical analysis

No statistical comparisons were performed as part of this study. Descriptive statistics including mean (standard deviation), median and proportions are reported.

Results

Clinical details of the patient cohort

Twenty patients were included in the study. Table 1 shows the clinical demographics of the patient cohort. The median age of the patients was 71 years (range 45–82). The cohort included 10 males and 10 females. Fifty percent of patients had been treated with rtPA prior to mechanical intervention. The majority of cases had an internal carotid artery and/or M1 occlusions (45% and 60% respectively). Several of the patients had occlusions in multiple sites (25%, data not shown). The average number of passes per patient was 2.35. Aspiration alone was used in 65% of cases and aspiration in combination with a stentriever device was used in the remaining 35% of patients. A thrombolysis in cerebral infarction score of 2b/3 was achieved in 90% of patients. To examine the possible underlying infection after the thrombectomy procedure, patient’s discharge summaries were reviewed. Sixteen patients had no infection present, one patient developed pneumonia (5%), one patient had a urinary tract infection (5%) and two patients had encephalopathy (10%).

Table 1.

Clinical details of acute ischemic stroke patients cohort.

		Number of patients
		(n = 20)	(%)
Age (years)
Median	71
Range	45–82
Gender
Male		10	50
Female		10	50
Site
ICA		9	45
M1		12	60
M2		4	20
ACA		1	5
rtPA
Yes		10	50
No		10	50
No. of passes
Mean	2.35
1		9	45
2		3	15
3		5	25
4		1	5
5+		2	10
Final TICI score
2a		2	10
2b		9	45
3		9	45
Source
Cardioembolic		11	55
Large artery		1	5
Unknown		6	30
Other		2	10
Devices used
Stentriever		0	0
Stentriever + aspiration		7	35
Aspiration only		13	65
Hemorrhagic conversion
Yes		4	20
No		16	80

ACA: anterior communicating artery; ICA: internal carotid artery; rtPA: recombinant tissue plasminogen activator; TICI: thrombolysis in cerebral infarction.

The universal bacterial gene 16S rDNA was not found in AIS clots

16S rDNA amplification was performed using both standard PCR and real-time qPCR. We used 50, 100 and 200 ng of gDNA template to study the expression of the 16S rDNA. Both standard PCR (Figure 1) and real-time qPCR (Figure 2) showed no expression of the 16S rDNA in any of the 20 clot samples analyzed. Amplification of the 16S rDNA was observed only in the positive control samples (Figures 1 and 2). Additionally, data from the Microbiome analysis showed absence of 16S material in clot samples of AIS patients; therefore, sequencing was not processed.

Figure 1.

Standard PCR results.

Representative PCR results of 16S rDNA run in 0.8% agarose gel. Lane 1: 1 Kb DNA ladder; lane 3: positive control (Escherichia coli); and lanes 2, 3–13 and 15–24: AIS patients’ clot samples. No expression of the 16S rDNA was observed in any of the AIS clot samples.

AIS: acute ischemic stroke; bp: base pair; PCR: polymerase chain reaction.

Figure 2.

Real-time qPCR results.

Representative 16S rDNA qPCR results of AIS patients. (a) qPCR results and (b) GAPDH results, showing the amplification and melt peak of positive control. No amplification was observed in clot samples (shown in red in (a)). (c) SYBR Green qPCR products run in 1.5% agarose gel electrophoresis. Lane 1: ladder; lanes 2 and 3: positive control; and lanes 4–23: AIS clot samples. (d) GAPDH runs in electrophoresis. No expression of the 16S rDNA was observed in the samples from AIS patients.

AIS: acute ischemic stroke; bp: base pair; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; qPCR: quantitative polymerase chain reaction.

Absence of bacteria in AIS clots by Gram staining

Gram staining was performed to confirm the absence of Gram-positive and Gram-negative bacteria in the AIS clots. S. aureus was used as the Gram-positive control and E. coli as the Gram-negative control (Figure 3(a) and (b), respectively). There was no evidence of Gram-positive or Gram-negative bacteria in the clot samples (Figure 3(c)).

Figure 3.

Gram staining results.

Gram stained images of Staphylococcus aureus, Escherichia coli and an AIS clot sample. (a) S. aureus Gram-positive was stained in a blue color (Gram stain, original magnification 100× oil) and (b) E. coli Gram-negative was stained in a red to pink color (Gram stain, original magnification 100× oil). (c) Neither Gram-positive nor Gram-negative bacteria were detected in the AIS clot samples (Gram stain, original magnification 100× oil).

AIS: acute ischemic stroke.

Discussion

Our study demonstrates the absence of bacterial signatures in the clot samples retrieved from stroke patients with large vessel occlusion (LVO), indicating that bacterial DNA, 16S rDNA, is not expressed in our AIS clot samples. Furthermore, the absence of bacteria in AIS samples is supported by Gram staining.

A number of studies have validated the notion that infection plays a role in vascular diseases such as atherosclerosis and thrombosis. Given the fact that atherosclerosis is one of the leading causes of AIS secondary to LVO, it seemed reasonable to hypothesize that infectious pathogens would be present in the clots of AIS patients as well. Bacteria that have been implicated in atherosclerosis include Helicobacter cinaedi, H. pylori, C. pneumoniae and Streptococci. Viruses which have been implicated in atherosclerosis include herpes simplex virus, Epstein-Barr virus, Coxsackie B virus and cytomegalovirus. In one recent study of thrombus aspirates in patients with ST-segment elevation myocardial infarction (STEMI), bacterial DNA from oral viridans streptococci were found in 78% of patients and periodontal pathogens in 35% of patients.¹⁶ It is suspected that during development of atherosclerotic plaques these bacterial populations contribute to smoldering inflammatory processes which contribute to plaque progression and eventually rupture. One key difference between the STEMI study and ours is the fact that acute myocardial infarction is usually due to local thrombosis on the surface of a plaque; however, most strokes secondary to LVO are due to embolus from cardiac thrombi or carotid atherosclerotic disease.¹⁷

Bacteria also can contribute to thrombosis in the absence of atherosclerosis. Studies in human and mouse blood and plasma have noted that spatial localization of Bacillus species affects the coagulation process and directly initiates the coagulation cascade when bacterial cells were in clusters.⁴ In addition, another study in human blood showed that bacterial species like S. aureus secrete pro-coagulant factors that can modulate immune reactions and promote clotting of plasma.¹⁸ Based on studies such as those mentioned above, we hypothesized that bacteria would be present inside the clots of AIS patients. However, we did not find any evidence of bacteria in any of the AIS clots analyzed.

It is important to point out that the absence of evidence is not necessarily evidence of absence. There are a few possible explanations for how bacteria could contribute to LVO without necessarily being found within the actual clot samples. One possible reason for the absence of the bacterial genes in our study may be possibly due to clot composition and formation. Several pro-coagulant proteins, including thrombin, fibrin, fibrinogen, von Willebrand factor and factor VIII, are increased during acute inflammatory events and infections.^19–23 Many of these factors, especially fibrin deposition, have been noted to play an important role in host antimicrobial defense.²⁴ In fact, fibrinogen or fibrin matrix has the inherent capacity to combat invading bacterial pathogens by entrapping bacteria, supporting the recruitment and activation of host immune cells, and thus limiting bacterial growth.²⁴ AIS clots are typically rich in fibrin/fibrinogen,^25–27 hence the absence of bacteria in the AIS clots may be due to the presence of fibrin components which act as antimicrobial agents against the growth of bacteria. Another possible reason is the fact that although bacteria may be found locally within atherosclerotic plaques and be involved in plaque rupture itself, they may not end up in the embolus which lodges in the intracranial artery.

Our study has limitations. First, our study has a relatively small sample size. Stroke in our North American population is usually due to embolic phenomenon from plaque rupture or cardiac sources. Thus, although bacteria were not found in the embolic clots, bacterial infection could have contributed to the source of the clot (i.e. bacteria could have been in the plaque which ruptured and caused the stroke embolus).

Conclusion

Our current study concludes that bacterial populations were not detected in the retrieved clots of AIS patients.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: the National Institutes of Health (grants R01NS105853 and R01NS076491).