Distinct Commensal Bacterial Signature in the Gut Is Associated With Acute and Long-Term Protection From Ischemic Stroke

Abstract

Background and Purpose- Commensal gut bacteria have a profound impact on stroke pathophysiology. Here, we investigated whether modification of the microbiota influences acute and long-term outcome in mice subjected to stroke. Methods- C57BL/6 male mice received a cocktail of antibiotics or single antibiotic. After 4 weeks, fecal bacterial density of the 16S rRNA gene was quantitated by qPCR, and phylogenetic classification was obtained by 16S rRNA gene sequencing. Infarct volume and hemispheric volume loss were measured 3 days and 5 weeks after middle cerebral artery occlusion, respectively. Neurological deficits were tested by the Tape Test and the open field test. Results- Mice treated with a cocktail of antibiotics displayed a significant reduction of the infarct volume in the acute phase of stroke. The neuroprotective effect was abolished in mice recolonized with a wild-type microbiota. Single antibiotic treatment with either ampicillin or vancomycin, but not neomycin, was sufficient to reduce the infarct volume and improved motorsensory function 3 days after stroke. This neuroprotective effect was correlated with a specific microbial population rather than the total bacterial density. In particular, random forest analysis trained for the severity of the brain damage revealed that Bacteroidetes S24.7 and the enzymatic pathway for aromatic metabolism discriminate between large versus small infarct size. Additionally, the microbiota signature in the ampicillin-treated mice was associated with a reduced gut inflammation, long-term favorable outcome shown by an amelioration of the stereotypic behavior, and a reduction of brain tissue loss in comparison to control and was predictive of a regulation of short-chain fatty acids and tryptophan pathways. Conclusions- The findings highlight the importance of the intestinal microbiota in short- and long-term outcomes of ischemic stroke and raises the possibility that targeted modification of the microbiome associated with specific microbial enzymatic pathways may provide a preventive strategy in patients at high risk for stroke. Visual Overview- An online visual overview is available for this article.

Keywords: ampicillin; bacteroidetes; metabolic pathways; microbiota; stroke; vancomycin.

FIG. 1. Broad-spectrum antibiotics modifies the bacterial density in feces and induces neuroprotection after stroke.

A) Mice received broad-spectrum antibiotics (AMNV) in the drinking water for 4 weeks (w4). AMNV treatment was discontinued 3 days before inducing MCAO or NMDA injection. Control mice (CTR) were age matched and received autoclaved water. Mice were sacrificed 3 days after MCAO induction (red) or 1 day after NMDA injection (blue). Stool pellets were collected as indicated. B) Body weight progression during AMNV treatment. X indicates mice which died during the AMNV treatment. C) Fecal bacterial density at several time points after the antibiotic supplementation was discontinued. D) Left, infarct volumetry in CTR and AMNV mice 3 days after MCAO induction. Right, lesion volumetry in CTR and AMNV mice 1 day after neocortical injection of NMDA.

FIG. 2. Acute AMNV-treatment is not protective from stroke and passive recolonization abolishes neuroprotection.

A) Left, mice received AMNV by gavage starting one day before MCAO surgery (d-1) until sacrifice (d+3) (“AMNV short”). Right, another group of mice received AMNV in the drinking water for 4 weeks following by the same treatment procedure as in the left panel (“AMNV long”). B) Left, bacterial density in feces before and 1 day to 3 days after AMNV treatment by gavage (AMNV short). Dash line indicates bacterial density in mice treated with AMNV in the drinking water for 4 weeks. Right, infarct volume in control mice not treated (CTR), after chronic AMNV treatment (AMNV long) and acute AMNV treatment (AMNV short) 3 days after MCAO induction. C) 4 weeks AMNV-treated mice were recolonized by co-housing for 2 weeks with a control wild-type mouse. Stool pellets were collected before and after recolonization. MCAO was induced after 2 weeks of co-housing. Mice were sacrificed 3 days after stroke and infarct volumetry was assessed. D) Left, bacterial density in feces 4 weeks after AMNV administration (black circles) and after 2 weeks of co-housing with a wild-type mouse (white circles). Dash line indicates bacterial density in wild-type mice. Right, infarct volume 3 days after MCAO in 4 weeks AMNV-treated mice and after recolonization.

FIG. 3. Single antibiotic treatment induces a distinct microbiota signature.

A) Shannon α-diversity index and B) richness and evenness of OTUs in control mice (CTR) and in mice treated with either neomcyin (N), vancomycin (V) or ampicillin (A) for 4 weeks. C) Relative abundances of bacterial families in fecal samples 4 weeks after the indicated antibiotic treatment. Each bar represents an individual mouse. D) Redundancy analysis ordination of the family composition across the three antibiotic treatment groups and controls. RDA1 and 2 explained 39% and 13% of the variance, respectively. For better clarity, phyla are indicated on the plot. Defined bacteria phyla are associated with specific antibiotic treatment groups and are distinct from the control group.

FIG. 4. Vancomycin or ampicillin alone is sufficient to improve acute stroke outcome.

A) Timeline of the single antibiotic administration. One day prior stroke mice were trained for the Tape Test. 3 days after stroke mice were tested for motor and sensory function by the Tape Test. B) Infarct volumes in antibiotic treated mice as compared to CTR mice 3 days after MCAO induction. C) Left, bacterial density in the feces 4 weeks after single antibiotic supplementation or in CTR mice. Dash line indicates bacterial density in mice treated with AMNV in the drinking water for 4 weeks. Right, correlation analysis of the fecal bacterial density with infarct volumes 3 days post MCAO after 4 weeks of single antibiotic treatment or in control mice. D) Sensorimotor function in CTR and antibiotic treated mice. Graphs show contact time (left) and time to remove the tape (right) from the contralateral forepaw 3 days after MCAO induction as relative to d-1.

FIG. 5. Single antibiotic treatment with vancomycin or ampicillin improves long-term stroke outcome.

A) CTR and 4-weeks antibiotic treated mice were tested for stereotypic behavior in the vicinity of a foreign mouse (intruder) by the open field test 14 days after stroke or sham surgery. Mice were sacrificed 35 days after MCAO induction for quantification of the brain tissue loss. B) Upper row shows a representative trajectory of mouse movements during the presence of the intruder mouse at the location indicated. Colored circle indicates the position of the mouse at the end of the 15 min trial. Lower row, quantification of the stereotypic behavior (time and counts) in the open field of CTR and antibiotic-treated mice in presence of the intruder 14 days after stroke and sham surgery. No statistical difference was observed between sham groups. C) Brain tissue loss was less pronounced in A-treated mice 35 days after MCAO as reflected by a significant decrease of ipsilateral volume loss compared to CTR mice.

FIG. 6. Association of specific bacteria and enzymatic pathways with infarct volume.

Random forest (RF) analysis showing predictive importance of A) Operational taxonomic units (OTU) at the family level (OTUs > 1% were included) and B) Enzymatic KEGG pathways trained for stroke outcome (size of the infarct volume) in all samples from the antibiotic treated- and control groups. “mse” (mean squared error) is a measure of the prediction accuracy of the RF as a function of permutating a variable. Variables associated with higher mse increase have higher predictive value. “Node purity increase” is a measure of how a split on a given variable reduces node impurity if applied to all nodes and all trees. Higher values are associated with more important variables. C) Heat-map of the significant regulated enzymatic pathways (rows) by samples (columns) after hierarchical clustering of treatment groups and KEGG pathways. Each column represents one mouse.