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.
Nature Medicine: doi:10.1038/nm.4194
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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
#
Nature Medicine: doi:10.1038/nm.4194
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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
KCTC 3501 strain NBRC
15882 or Lactobacillus
apodemi strain : ASB1
98 0.0219
22
AY991429
5.87
Lactobacillus animalis
KCTC 3501 strain NBRC
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
Nature Medicine: doi:10.1038/nm.4194
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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
48
# % ID to known species too low to imply taxonomy & No proofs of pathogenicity
Nature Medicine: doi:10.1038/nm.4194
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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).
Nature Medicine: doi:10.1038/nm.4194
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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)
Goat 1:1000 Abcam (Cambridge, UK)
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)
Goat 1:500 Abcam (Cambridge, UK)
ZO-1 Rabbit 1:1000 Invitrogen (California, USA)
Nature Medicine: doi:10.1038/nm.4194
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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)
Donkey anti-rabbit Alexa Fluor 594 1:500 Jackson ImmunoResearch
(Pennsylvania, USA)
Donkey anti-rabbit Alexa Fluor 647 1:500 Jackson ImmunoResearch
(Pennsylvania, USA)
Donkey anti-sheep Alexa Fluor 488 1:500 Jackson ImmunoResearch
(Pennsylvania, USA)
Nature Medicine: doi:10.1038/nm.4194
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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.
Nature Medicine: doi:10.1038/nm.4194
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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.
Nature Medicine: doi:10.1038/nm.4194
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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.
Nature Medicine: doi:10.1038/nm.4194
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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.
Nature Medicine: doi:10.1038/nm.4194