Translocation and dissemination of commensal bacteria in ...Translocation and dissemination of...

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Translocation and dissemination of commensal bacteria in post-stroke infection

Dragana Stanley 1, Linda J Mason 2, Kate E. Mackin 3, Yogitha N. Srikhanta 3, Dena Lyras 3,

Monica D. Prakash 4, Kulmira Nurgali 4, Andres Venegas 5, Michael D. Hill 5, Robert J.

Moore 3,6 and Connie H. Y. Wong 7,*

1. School of Medical and Applied Sciences, Central Queensland University, Australia;

2. Department of Biochemistry, Monash University, Australia;

3. Infection and Immunity Program, Monash Biomedicine Discovery Institute and

Department of Microbiology, Monash University, Australia;

4. Centre for Chronic Diseases, College of Health and Biomedicine, Victoria University,

Australia;

5. Stroke Unit, Department of Clinical Neurosciences & Hotchkiss Brain Institute,

University of Calgary, Alberta, Canada;

6. School of Science, Royal Melbourne Institute of Technology University, Australia;

7. Centre for Inflammatory Diseases, Department of Medicine, School of Clinical

Sciences, Monash University, Australia.

SUPPLEMENTARY INFORMATION

Supplementary Tables 1-4

Supplementary Figures 1-13

Nature Medicine: doi:10.1038/nm.4194

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Supplementary Table 1: Baseline characteristics of the enrolled subjects.

Characteristics Stroke without infection Stroke with infection Hospital Healthy

Number of patients, no. (%) 28 (77.8) 8 (22.2) 0 (0) 0 (0)

Age, years, median (IQR) 65.5 (56.5, 72.5) 76.5 (72.75, 83.5) 71.0 (47.0, 83.0) 63.0 (43.75, 79.5)

Male sex, no. (%) 16 (57.1) 6 (75) 5 (55.6) 5 (50%)

Mortality, no. (%) 2 (7.1) 3 (37.5) 0 (0) 0 (0)

NIH Stroke Scale, median (IQR) 6.5 (4, 10.75) 8 (4.25, 20.5) - -

Localisation of infarction, no. (%)

MCA 16 (57.1) 6 (75) - -

ACA 1 (3.6) 0 (0) - -

PCA 3 (10.7) 0 (0) - -

Cerebella 1 (3.6) 0 (0) - -

Pontine/Midbrain 4 (14.3) 0 (0) - -

Multiple territories 3 (10.7) 2 (25) - -

Hemispheric, no. (%)

Left 16 (57.1) 4 (50) - -

Right 8 (28.6) 1 (12.5) - -

Bilateral 0 (0) 2 (25) - -

Stem 3 (10.7) 0 (0) - -

Shower 1 (3.6) 1 (12.5) - -

Infection site, no. (%)

Chest

-

2 (25)

-

-

Urinary tract - 6 (75) - -

Mortality from infection, no. (%)

Chest - 2 (100) - -

Urinary tract - 1 (16.7) - -

The National Institute of Health (NIH) Stroke Scale is composed of 11 items to use as a tool by clinicians to objectively quantify the stroke

impairment.

ACA, anterior cerebral artery; MCA, middle cerebral artery; PCA, posterior cerebral artery; IQR, interquartile range; SD, standard deviation.


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Supplementary Table 2: Elevated relative abundance of OTUs found in post-stroke lung

microbiotas.

OTU Fold

increase in

post-stroke

lung

Taxonomy (Blastn against

16s Microbial database)

%ID p value

(Kruskal-

Wallis)

Ref.

OTU635685 5.83 Actinomyces europaeus

strain CCUG 32789A

96 0.0032 43

AY990983

7.96

Lactobacillus animalis

KCTC 3501 strain NBRC

15, Lactobacillus apodemi

strain : ASB1 16S

100 0.0054

22

OTU475344

6.81

Clostridium xylanolyticum

strain DSM 6555 or

Clostridium

saccharolyticum WM1

strain WM1

93 0.0073

44

DQ447846

9.78

Lactobacillus aviarius 16S

or Lactobacillus salivarius

strain HO 66

>96 0.0093

22

AB117565

7.94

Bacteroides massiliensis

strain B84634 16S,

Bacteroides finegoldii DSM

17565 strain JCM 13345

>95 0.0094

45

EU470613

4.44

Twelve Escherichia and

Shigella strains, all with

99%ID to E. fergusonii, E.

coli, S. sonnei, S. flexneri, S.

boydii and S. dysenteriae

species

99 0.01

46,47

EU507486

17.04

Akkermansia muciniphila

ATCC BAA-835 strain

ATC BAA-835

91 0.0124

#

OTU13194

Stroke only Clostridium glycolicum

strain DSM 1288,

Clostridium bartlettii DSM

90-91 0.0209

#


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16795 or Clostridium

difficile 630 strain 630

OTU100748

Stroke only More than 20 strains

belonging to Staphylococcus

species with %ID of 93-94

to strains of S. cohnii, S.

haemolyticus, S. caprae, S.

gallinarum, S. capitis , S.

xylosus, S. aureus, S.

saprophyticus, S.

epidermidis and others.

>93 0.0209

48

OTU103222

Stroke only Bifidobacterium longum

NCC2705 strain NCC2705

91 0.0216

#

OTU103823

Stroke only Barnesiella intestinihominis

YIT 11860 strain YIT

11860

85 0.0219

#

EU451393

Stroke only Lactobacillus animalis


15882 or Lactobacillus

apodemi strain : ASB1

98 0.0219

22

AY991429

5.87

Lactobacillus animalis


15882 or Lactobacillus

apodemi strain : ASB1

99 0.0240

22

FJ879596

17.78

Clostridium indolis strain 7

or Clostridium

saccharolyticum WM1

strain WM1

94-95 0.0258

44

EU460011

8.89

Eleven strains of

Streptococcus, all with

98%ID to the species of: S.

infantarius, S. lutetiensis, S.

equinus, S. luteciae, S.

pasteurianus, S. gallolyticus

and S. macedonicus

98 0.0286

49

HQ740469

13.33

Clostridium

saccharolyticum WM1

strain WM1, Clostridium

celerecrescens strain DSM

>96 0.0290

44


5

5628, Clostridium

xylanolyticum strain DSM

6555, Clostridium indolis

strain 7 or Clostridium

algidixylanolyticum strain

SPL73

OTU182

15.56

Allobaculum stercoricanis

DSM 13633 (followed by

multiple species of

Eubacterium at 87%ID)

90 0.0290

#

DQ014676

2.86

Ruminococcus albus 7 strain

7,

Hydrogenoanaerobacterium

saccharovorans strain

SW512 or Clostridium

methylpentosum DSM 5476

strain R2

92 0.0342

#

ADWV01000001

4.81

Twelve strains of

Escherichia or Shigella

species all with 97%ID,

including E. fergusonii, E.

sonei, E. coli, E. albertii, S.

sonnei, S. flexneri, S.

dysenteriae and S. boydii

97 0.0443

46,47

AB008513

12.89

Brevundimonas nasdae

strain W1-2B,

Brevundimonas vesicularis

strain Busing

100 0.0443

50,51

DQ806292

7.78

Akkermansia muciniphila

ATCC BAA-835

96 0.0450

&

AB233334

22.22

Staphylococcus sciuri

subsp. rodentium strain

GTC 844

100 0.0475

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# % ID to known species too low to imply taxonomy & No proofs of pathogenicity


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Supplementary Table 2 References

22. Martinez, R.M., Hulten, K.G., Bui, U. & Clarridge, J.E., 3rd. Molecular analysis and clinical

significance of Lactobacillus spp. recovered from clinical specimens presumptively

associated with disease. Journal of clinical microbiology 52, 30-36 (2014).

43. Mabeza, G.F. & Macfarlane, J. Pulmonary actinomycosis. The European respiratory

journal : official journal of the European Society for Clinical Respiratory Physiology 21,

545-551 (2003).

44. Kuhn, J.H. Qualitative and Quantitative Assessment of the “Dangerous Activities”

Categories. (ed. Maryland, C.f.I.a.S.S.a.) (2005).

45. Schreiner, A. Anaerobic pulmonary infections. Scand J Gastroenterol Suppl 85, 55-59

(1983).

46. Bennish, M.L. Potentially lethal complications of shigellosis. Reviews of infectious diseases

13 Suppl 4, S319-324 (1991).

47. Hill, R.B., Jr., Rowlands, D.T., Jr. & Rifkind, D. Infectious pulmonary disease in patients

receiving immunosuppressive therapy for organ transplantation. 1964. Reviews in medical

virology 9, 5-10; discussion 13-15, 10-14 (1999).

48. Gillet, Y., et al. Association between Staphylococcus aureus strains carrying gene for

Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young

immunocompetent patients. Lancet 359, 753-759 (2002).

49. Beck, M., Frodl, R. & Funke, G. Comprehensive study of strains previously designated

Streptococcus bovis consecutively isolated from human blood cultures and emended

description of Streptococcus gallolyticus and Streptococcus infantarius subsp. coli. J Clin

Microbiol 46, 2966-2972 (2008).

50. Shang, S.T., et al. Invasive Brevundimonas vesicularis bacteremia: two case reports and

review of the literature. J Microbiol Immunol Infect 45, 468-472 (2012).

51. Chi, C.Y., Fung, C.P., Wong, W.W. & Liu, C.Y. Brevundimonas bacteremia: two case

reports and literature review. Scand J Infect Dis 36, 59-61 (2004).


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Supplementary Table 3: Primary antisera and dilutions for Immunohistochemistry.

Tissue antigen Host species Dilution Source

β-Tubulin III Chicken 1:1000 Abcam (Cambridge, UK)

Choline Acetyl Transferase (ChAT) Goat 1:200 Millipore (Temecula,

Canada)

Glial fibrillary acidic protein

(GFAP)

Goat 1:1000 Abcam (Cambridge, UK)

Neuronal Nitric Oxide Synthase

(nNOS)


Neuropeptide-Y (NPY) Sheep 1:1000 Millipore (Temecula,

Canada)

S100β Rabbit 1:1000 Abcam (Cambridge, UK)

Tyrosine Hydroxylase (TH) Sheep 1:1000 Millipore (Temecula,

Canada)

Vasoactive Intestinal Peptide (VIP) Rabbit 1:1000 Abcam (Cambridge, UK)

Vesicular Acetylcholine Transporter

(VAChT)


ZO-1 Rabbit 1:1000 Invitrogen (California, USA)


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Supplementary Table 4: Secondary antisera and dilutions for Immunohistochemistry.

Antiserum Dilution Source

Donkey anti-chicken Alexa Fluor 594 1:500 Jackson ImmunoResearch

(Pennsylvania, USA)

Donkey anti-goat Alexa Fluor 488 1:500 Jackson ImmunoResearch

(Pennsylvania, USA)

Donkey anti-rabbit Alexa Fluor 488 1:500 Jackson ImmunoResearch

(Pennsylvania, USA)


(Pennsylvania, USA)


(Pennsylvania, USA)

Donkey anti-sheep Alexa Fluor 488 1:500 Jackson ImmunoResearch

(Pennsylvania, USA)


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Supplementary Figure 1: Comparable infarct volume and gut permeability between post-

stroke SPF and GF mice. (a) Quantification of the infarct size of post-stroke SPF and GF mice at

24 h. n = 5 mice per group; error bars, SEM; NS denotes not statistically significant by unpaired

two-tailed Student’s t test. (b) Quantitative analysis of FITC-dextran translocation determined of

sham-operated or post-stroke SPF and GF mice at 3 h post-stroke. n = 5 mice per group; error bars,

SEM.


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Supplementary Figure 2: Stroke induce no changes in lung microbiota alpha diversity. There

was no difference in alpha diversity measures between post-stroke and sham mice lung detected

microbiota in either Richness (P = 0.73), Evenness (P = 0.81), Shannon (P = 0.89) or Simpson (P =

0.45) index.; n = 5 per group; error bars, SEM; performed in technical duplicates; unpaired two-

tailed Student’s t test.


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Supplementary Figure 3: OTUs that are shared and unique to sham-operated and post-stroke

lung microbiota. Venn diagram showing shared (green) and unique OTUs between microbial

communities within the lungs of sham-operated mice (blue) and post-MCAO mice (red). n = 5 per

group, performed in technical duplicates.


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Supplementary Figure 4: Post-stroke gut microbial biomass. Quantitative analysis of microbial

biomass in the duodenum (a) and jejunum (b) expressed as the ratio of copy numbers between the

16S and Rpp30 genes; n = 7 mice per group; error bars, SEM; NS denotes not statistically

significant by unpaired two-tailed Student’s t test.


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Supplementary Figure 5: Group-to-group similarities between microbial communities in the

various tissue of sham-operated and post-stroke mice. Weighted UniFrac sample-to-sample

distance matrix calculated in QIIME was used to analyse ANOSIM pairwise group-to-group


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similarities between microbial communities in the various tissue of sham-operated (a) and post-

stroke (b) mice at 24 h. ANOSIM matrix is presented as 3D Principal Coordinate Analysis plot. n =

5 mice per group.


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Supplementary Figure 6: Vascular permeability post-stroke. Quantitative analysis of Evans

blue extravasation in the lung (a), duodenum (b) and colon (c) at 3 and 24 h after MCAO. n = 7

mice per Sham and 3 h group, n = 10 per 24 h group; error bars, SEM.


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Supplementary Figure 7: Representative vertical slices of small intestine contractile motility

in sham-operated and post-MCAO mice. Vertical slices were taken at the proximal (oral) and

distal (anal) ends at 24 h post-surgery and corresponding 1 minute sections of each slice were

quantified for number and size (amplitude) of contractions.


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Supplementary Figure 8: Goblet cells numbers post-stroke. Quantification of goblet cell

numbers in the duodenum (a) and colon (b) of sham-operated and post-stroke mice in a double-

blinded manner. n = 8 mice per group; error bars, SEM.


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Supplementary Figure 9: Gating strategy to enumerate mesenteric lymph node leukocyte

populations. Gating strategy of flow cytometric analysis of leukocyte subpopulations in the

mesenteric lymph nodes in sham-operated and post-MCAO mice at 3 and 24 h after surgery.


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Supplementary Figure 10: Mesenteric lymph node leukocyte populations. Quantification of the

number of total CD4+ T cells (a), CD4- T cells (b) and CD103+ cells (c) in MLN after stroke. n = 9

mice per 3 h group, n = 7 mice per 24 h group; error bars, SEM.


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Supplementary Figure 11: Neuronal changes in the gut after stroke. Stroke induced changes in

the proportion of TH+ neuron (a) and fibres (b), as well as the proportion of NPY+ neuron (c) within

the submucosal plexus of the ileum was quantified. n = 5 mice per group; error bars, SEM; NS

denotes not statistically significant by unpaired two-tailed Student’s t test.


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Supplementary Figure 12: Bacteriological analysis of lymph nodes post-stroke. Quantitative

analysis of bacterial loads from accessary axillary lymph nodes (AALN, a), mediastinal lymph

nodes (MSLN, b) and mesenteric lymph nodes (MLN, c) of sham-operated and post-MCAO mice

treated with saline or propranolol (PPL). Data are presented as number of colony forming units

(CFU) per g of tissue. n = 6 mice per Sham group, n = 10 mice per MCAO group; error bars, SEM;

NS denotes not statistically significant by unpaired two-tailed Mann Whitney’s u test.


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Supplementary Figure 13: Examination of aspiration after stroke. FITC-dextran was

administrated intranasally on sham-operated and post-MCAO mice at reperfusion, the serum and

lungs were collected to assess aspiration. n = 9 mice per group; error bars, SEM; **P < 0.01, NS

denotes not statistically significant versus Sham by unpaired two-tailed Student’s t test.


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