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Bacterial Spores as Probiotics: Mode of Action
Professor Simon Cutting School of Biological Sciences Royal Holloway University of London Egham, Surrey TW20 0EX, UK [email protected]
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Probiotech 2
1. Sporulation in Bacilli
2. Use of Bacillus spores as probiotics
3. How do they work?
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Plate colony Phase Microscopy Electron microscopy Atomic Force microscopy
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Sporulation Vegetative Growth
Life cycle of Bacillus species
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200 nm
A
100 nm
B
200nm
C
100 nm 200 nm 0,5 µm
F E D
Spores of different isolates of B. subtilis
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Spores are very robust
Science (1995). 268: p1060-4
Resistance to UV irradiation Temp 65oC-80oC Extreme heat-short term (<8 min) to 235oC Resistance to solvents and noxious chemicals
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Use As Probiotics, Dietary Supplements and Nutrafoods
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Dietary supplements, food supplements B. subtilis, B. cereus, B. coagulans (aka Lactobacillus sporogenes), B. clausii
•GanedenBC30. Spores of B. coagulans added to muffins and baked foods, alcohol, chocolate in the USA etc.
•B. subtilis has registration for food use in the UK (2007) and Italy (<1991)
•B. coagulans has GRAS status in the USA (2008)
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Natto B. subtilis var. Natto Japanese staple
BioPlus 2B Spores of B. subtilis & B. licheniformis Animal feed Christian Hansen (Denmark)
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Main Street Gourmet (USA) obtained from Isabella’s Healthy Bakery “Activate” Probiotic muffins impregnated with spores
Turtle Island Soup (USA) “Souper Food” Soups fortified with spores
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1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
1.E+09
1.E+10
1 2 3 4 5 6 7 8
HU58
Protexin
Control
Before cooking
0 7 14 28 42 56 84
Days
Spo
res
cou
nt
/ gr
am
Survival of spores incorporated in biscuits (McVities) after 84 days
These are background counts And are Bacillus spores present in your biscuit mix. The biscuit mix is not sterile and data shows that cereals, wheat etc have a basal level of spore counts.
Starting CFU HU58 Tube 1 + 1kg mix = 6.57 X 108 spores/g PXN21 Tube 1 + 1kg mix = 1.33 X 109 spores/g
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Pigmented GIT isolates of B. indicus
Vegetative Growth Spores
Vegetative Cells Sporulation
13
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A.
B.
C.
P1-492 nm
l max
3
P2-467 nm
l max P3-455 nm
l max
P3-455 nm
l max
Retention time (minutes) Ab
sorb
ance
(ar
bit
rary
un
its)
450nm
450nm
450nm
GB1
AU
0.000
0.010
0.020
Minutes
20.00 40.00 60.00
Retention time (minutes)
SF223
HU28
PY79
450nm
0.00
0.10
0.20 D.
Initial Carotenoid Characterisation
14
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0m 20m 60m
100 50
% C
aro
ten
oid
HU36 cells incubated with simulated gastric fluid (SGF) Carotenoids first extracted from HU36 spores and then incubated in SGF
b-carotene as a control
Bacillus Carotenoids are Gastric Stable
15 Probiotech
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5.2 5.3
1.0
1.9
0.2
2.0
0.50.4
0
1
2
3
4
5
6
7
8
pure lutein pure
β-carotene
pure lycopene pure astaxanthin purified HU36
carotenoids
carotenoids in
HU36 lyophilized
bacteria
purified GB1
carotenoids
carotenoids in
GB1 lyophilized
bacteria
Bio
ava
ila
bility (
%)
D I E T
A G R I C U L T U R E
E N V I R O N M E N T
IN VIVO BIOAVAILIBILITY RESULTS
• Bioavailability = % of carotenoids recovered in plasma + liver + adipose tissue in rats
β-carotene
plasma liver adipose tissue
lycopene
plasma liver adipose tissue
lutein
plasma liver adipose tissue
astaxanthin
plasma liver adipose tissue
HU36 carotenoids
plasma liver adipose tissue
carotenoids in HU36 cells
plasma liver adipose tissue
GB1 carotenoids
plasma liver adipose tissue
carotenoids in GB1 cells
plasma liver adipose tissue
HU36 carotenoids
plasma liver adipose tissue
Bacterial carotenoids are bioavailable (HU36>GB1)
mainly recovered in liver and adipose tissue
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CONCLUSIONS
Bacterial carotenoids are
- more stable than standard carotenoids
(towards dietary iron in micellar system)
- better antioxidants
than standard carotenoids
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QPS Species of Bacillus that carry Carotenoids Bacillus megaterium (Yellow/Orange) and Bacillus pumilus (red) • Use for prevention of CardioVascular Disease (CVD) • Potential anti-cancer agent • Iron deficiency since carotenoids enhance iron uptake
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Bacillus species are gut commensals
Found in the human gut at ~104 spores/g of feces Recovered from ileal biopsies 30% of the gut flora are spore formers Most Bacilli produce bile salt hydrolase Some species have evolved into pathogens (B. anthracis and B. cereus) Can persist for up to 1 month in the gut of the mouse
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Analysis of Spore Counts in Faeces Shows More Spores Excreted than Inoculated by a Factor of 3-6
No
. of
hea
t-re
sist
ant
spo
res
in f
aece
s
Individual mice given single dose of spores and shed heat-resistant cfu determined in faeces
20 Probiotech
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Foreign gene Bacillus gene
559 bp
466 bp
RtetC
5
RtetC
5
FcotB
1
FcotB
2
Germination, proliferation and sporulation in the GI-tract
1) Construction of chimeric gene, expressed during germination/growth or during sporulation
2) Dose mice with spores, extract mRNA and quantify expression
I I I
C C C I, ileum, C, colon 21 Probiotech
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Mouse Duodenum Jejunum Ileum Large int. Duodenum Jejunum Ileum Large int. Duodenum Jejunum Ileum Large int.
1 - ++ - - - ++ - - - + - -
2 - ++ + - - ++ - - - + - -
3 - ++ - - - ++ + - - + - -
4 - ++ - - - ++ - - + - -
Naïve - - - - n/a n/a n/a n/a n/a n/a n/a n/a
1 - - - - - - - - - - + +
2 - - - - - - - - - - + +
3 - - - - - - - - - - + +
4 - - - - - - - - - - + +
Naïve - - - - n/a n/a n/a n/a n/a n/a n/a n/a
Mouse Duodenum Jejunum Ileum Large int. Duodenum Jejunum Ileum Large int.
1 - - - - - - - -
2 - - - - - - - -
3 - - - - - - - -
4 - - - - - - - -
Naïve n/a n/a n/a n/a - - - -
1 - - + + - - + ±
2 - - + + - - + ±
3 - - + + - - + ±
4 - - + + - - + ±
Naïve n/a n/a n/a n/a - - - -
Sporulation
48h
Germination
Sporulation
Germination
18h 24h 30h
36h
Approx. 10-16% of spores germinate and then re-sporulate in the GI-tract
Measure germination by using a vegetative Bacillus gene chimera Measure re-sporulation by using a sporulation Bacillus gene chimera
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Fate of Spores
* Casula & Cutting. 2002. Appl Environ Microbiol 68:2344-52
** Hoa et al. 2001. Appl Environ Microbiol 67:3819-23
*** Duc et al. 2004. Vaccine 22:1873-85
germinate *
re-sporulate **
taken up by cells of
lymphoid tissues
e.g. M-cells of Peyer’s patches
replicate **
germinate ***
23 Probiotech
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Anti-spore labelling
Anti-b-Gal labelling (vegetative gene expression*)
45m 2h 5h 24h
45m 2h 5h 24h
Phagocytosis of rrn0-lacZ spores by macrophage-like cell line, RAW264.7
Ingestion and prolonged survival could induce CTL responses
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Stomach
Spores survive intact
vegetative Bacillus killed
Small Intestine
Nutrient rich
Spore germination &
dissemination to GALT
Colon
Re-sporulation
GIT
Excretion of spores
into the soil
Symbiotic interactions
with plants
Consumption of
plants and spores
spores
vegetative cells
Bacillus as a gut commensal Commonly found in the human GI-tract at 1 X 104/g
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BS3510 Lecture 7 26
Mechanisms for Probiosis 1) Stimulation of innate immunity
2) Synthesis of antimicrobials that kill pathogens 3) Adsorption of toxins in the GI-tract 4) Rebalancing the gut microflora
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Innate Immunity
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b-actin
TNFa
IFNg
0
20
40
60
80
100
120
140
160
180
200
0 100 200 300
Hours after inoculation %
expre
ssio
n
Cytokine mRNA in vivo
IFN-g Th1 response Cellular immunity
& IgG2a
IFN-g
TNF-a
1) Immune Stimulation
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Evidence for Innate Immunity and Induction by Non-Recombinant Spores carrying no antigen
1. C. difficile infection
Naive
PY79
rA26
-39+
rB15
-24
PP108
PP14
2
rA26
-39+
rB15
-24
40
50
60
Day 15
o.g. i.p.
Su
rviv
al a
fte
r in
fectio
n (h
ou
rs)
o.g. i.p.
Day 15
60
50
40
Surv
ival
aft
er in
fect
ion
(h
ou
rs)
Naive PY79 rA26-39 + rB15-24
PP108 PP142 rA26-39 + rB15-24
29
Vaccination experiment Shaded box shows increased survival of hamsters pre-dosed with B. subtilis PY79 spores and then challenged with C. difficile
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In this study the spores were delivered via the intra-peritoneal (i.p.) route to provide protection against an i.p. challenge with B. anthracis STI spores. The spores were administered to mice 48h and 24h prior to challenge with B. anthracis (approx 100 MLD) and protection afforded against anthrax was monitored. Results showed that pre-exposure administration of the B. subtilis spores was able to provide partial protection against anthrax infection in mice (expt done at Porton Down).
30
2. Anthrax
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Survival rates
1 2 3 4 5 6 7 8 9 10 11 12 13 14 0
120
100
80
60
40
20
0
Days post challenge
Su
rviv
al
(%)
27 days
Challenge with live H5N2
Day 1/i.n. Day 14/i.n.
No antigen-immunotherapy
3. Influenza
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0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
10^6 10^7 10^8 10^6 10^7 10^8 1ng 10ng 20ng 10^7 Mediaonly
OD
65
5
106 107 108 106 107 108 1ng 10ng 20ng 107 media
PY79K latex beads LPS VC
Induction of innate immunity. Spores stimulate NF-Kb pathway via interaction with Toll-like receptors
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Clostridium difficile Infection (CDI)
Major nosocomial pathogen ~4,000 deaths per annum in the UK No vaccine Protective antigen thought to be toxin A and toxin B All vaccines under development are parenteral Emergence of the 078 ribotype from pigs to humans
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In ~30% of patients, CDI can return in 2-3 weeks (relapse or recurrence)
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Mice dosed every day for 7 days pre infection or every day post infection Kaplan-Meier survival plot for mice infected with C. difficile VPI 10463. Groups received oral doses of live B. subtilis PXN21 spores as treatment either pre or post infection. Post infection slightly better
Effect on C. difficile infection, pre and post dosing
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Pre-dosing effect is likely to be due to innate immunity Post-dosing effects could be due to subtraction of toxin the GI-tract?
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Adsorption to Spores
toxin B. subtilis spores -Net negative charge -hydrophobic
+
Ionic and hydrophobic interactions
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Toxin A
Toxin B
In vitro binding of toxin to spores Caecal extracts from post-dosed mice
Toxin A Toxin B
1 caecum extracts post-dosing 2 caecum extracts no spores 3 caecum extracts no C. difficile 4 purified toxins 5 = toxin detected in supernatants of caecal extracts
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Secretion of beneficial molecules in the GI-tract Most Bacilli produce Nattokinase, a protease that reduces blood clotting Antimicrobials. Depends on species and strain but, Plipastatin, Bacilysin, Subtilosin, Fengycin, Iturins, etc Many strains can be administered together with antibiotics and this allows temporary restoration of the gut microflora during prophylaxis
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Spores, and Specifically Sporulation are Important for Development of the GALT
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Summary Gut commensals Spore probiotics are ‘live’ entities that germinate in the GI-tract, proliferate and then re-sporulate They promote a healthy GALT Stability They have unique resistance properties enabling them to be stored at room temperature indefinitely Efficacy Proven by worldwide use Emerging clinical trial data Mechanism Stimulate innate immunity Somehow promote GALT development Adsorption of toxins Secretion of antimicrobials
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Phase 1/2a clinical trial 2013
www.cdvax.org
Dr. Hong Huynh Siamed Hasseni Dr. Irene Bianconi Saba Anwar Dr. Lluis Sempere William Ferreari Dr. Stephanie Willing Celia Rodriqgues Dr. Anil Chandrashekran Emma Popescu Ms. Krisztina Hitri Jacob Goonesana Ms. Nicola Byrne Alex Curilovs
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Kaplan-Meier survival plot for mice infected with C. difficile VPI 10463. Groups received oral doses of live B. subtilis PXN21 spores post infection at a concentration of either 109 or 107 spores per dose.
Suppression of C. difficile infection is not dose dependent
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Live spores rather killed spores are needed for suppression of C. difficile infection
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pH
% H
ydro
ph
ob
icit
y z
(mV
)
100
80
60
40
20
0
1 0 3 2 5 4 7 6 8
0
-20
-40
-60
-80
At all pH values spore is negative charged, but as pH drops below pI protein becomes + charged At all pH values, spore is hydrophobic and thus able to interact with hydrophobic molecules such as LPS, virions, etc
hydrophobicity
SURFACE CHARGE zeta potential