Physiology of EHEC carriage by ruminants
Antagonist properties of probiotics
Objectives
Limiting the risk of food-borne infections
Limiting EHEC shedding by ruminants: reduction of
EHEC carriage
Knowledge of physiology
and ecology of EHEC in the
bovine gastro-intestinal tract
Selection and evaluation
of probiotic strains
Effects of probiotics on EHEC survival in ruminant
digestive contents
 Probiotics can positively modulate the microbial balance of
the animal gut and thereby reduce its colonisation by foreign
bacteria
 Some of them may also exert direct inhibitory effects on
foreign bacteria (competition for adhesion/nutrients,
bacteriocins, inhibitory compounds…)
Method
3 sheep fed hay +
wheat
3 sheep fed
3 sheep fed
9 CFU
hay
+
wheat
+
5
x
10
hay + wheat + 4 x 109 CFU
Lactobacillus acidophilus
Saccharomyces cerevisiae I-1077
BT-1386
Rumen contents through canula (pH 5.7)
Abomasum contents after feeding (pH 2.5) at slaughter
Jejunal and caecal contents at slaughter (pH 7.8 and 7.7 respectively)
Incubation of STEC strains at 39°C
EDL933 (O157:H7), 4 acid-resistant, 4 acid-sensitive strains (diverse serotypes)
Consequences of acid-resistance in STEC
Adaptation to acid stress
Induction of acid-resistance
mechanisms
- to acid stress in the bovine abomasum (pH 2.5)
Resistance
- to food processing
- to acid stress in the human stomach
Increases the risk of food poisoning in humans
% survival
STEC survival after 24 h in the rumen fluid
Anaerobiosis
50
40
In the presence of S. cerevisiae or L.
acidophilus (3 x 105 CFU/ml), the
survival rates dropped below 1.48%.
30
20
10
0
EDL933
AR1
AR2
AR3
AR4
AS1
AS2
AS3
Acid-sensitive STEC
Log cfu/ml
Acid-resistant STEC
7
6
Log cfu/ml
AS4
7
6
5
5
4
4
3
3
EDL933
AR1
EDL933
2
2
1
EDL933 + L. acidophilus
1
0
0
0
2
4
6
8
10
12
14
16
18
20
Time (h)
EDL933 persists 12 h in the rumen fluid,
then declines. Mortality is observed at 18 h.
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (h)
Mortality occurred earlier in the
presence of L. acidophilus
Chaucheyras Durand et al. 2010, AEM
STEC survival after 2 h in the abomasal fluid
Survival (%)
Static culture
100
90
80
70
60
50
40
30
20
10
0
EDL933
AR1
AR2
AR3
AR4
Acid-resistant STEC
AS1
AS2
AS3
AS4
Acid-sensitive STEC
EDL933 and acid-resistant strains survive in the abomasal contents at variable levels.
Acid-sensitive strains show high mortality rates
Chaucheyras Durand et al. 2010, AEM
Induction of acid-resistance in the rumen contents
STEC 106 cfu/ml
6h
Rumen fluid
Abomasal fluid
Acid-sensitive strains become acidresistant after transit in the rumen
fluid whatever the diet
6
Log cfu/ml
Survival
4
2
0
0
AR1
AS2
EDL933
1
2
AR1P
AS2P
EDL933 P
3
4
Time (h)
Transit of STEC strains in the cattle
gastro-intestinal tract may increase the
risk of human infection
Chaucheyras Durand et al. 2010, AEM
STEC growth after 2 h in jejunal contents
Static cultures
8,00
t=0h
t=2h
Log cfu/ml
7,00
6,00
Significant growth of STEC
strains in both intestinal
contents
5,00
4,00
EDL933
AR1
AR2
AR3
AR4
AS1
Acid-resistant STEC
AS2
AS3
AS4
Acid-sensitive STEC
STEC growth after 8 h in caecal contents
9
Log cfu/ml
8
Probiotics did not limit
STEC growth in these
compartments
t=0h
t=8h
7
6
5
4
EDL933
AR1
AR2
AR3
Acid-resistant STEC
AR4
AS1
AS2
AS3
Acid-sensitive STEC
AS4
Chaucheyras Durand et al. 2010, AEM
EHEC O157:H7 growth after 24 h in faecal suspension
3,50
Control without L.A.
L.A.
2,50
1,50
0,50
-0,50
-1,50
-2,50
-3,50
+ L.A.
3.4 x 105.ml-1
+ L.A.
3.2 x 106.ml-1
+ L.A.
8.5 x 107.ml-1
Dose-dependent inhibition of STEC growth by L. acidophilus
Chaucheyras Durand et al. 2006, AEM
Summary
 Acid-resistance confers an ecological advantage to STEC strains for their persistence in
the rumen
 Acid sensitive strains can become resistant after a short transit in rumen contents
 The rumen is a critical compartment controlling STEC development
 STEC are able to grow significantly in intestinal and fecal contents
 Probiotics used in this in vitro study induce delayed STEC mortality but only in rumen
contents, or high concentrations are required
Need for selection of more efficient probiotic strains:
Acting more quickly in the rumen
 Efficient in the hindgut at lower concentration
 Not inducing acid resistance mechanisms
 Targeting STEC strains
Deep knowledge of ecology and physiology of STEC
in the bovine digestive tract is required
Effect of the diet, autochtonous flora, and oxygen on EHEC growth
Rumen contents, anaerobiosis
pH
10
t0
t24
9
Log10 CFU/ml
8
Hay
7
Acetate concentration
6.65
48.25 mM
Hay+Wheat 5.37
71.80 mM
6
5
4
3
2
1
0
Hay
Hay + Wheat
Growth when animals were fed hay
Mortality when animals were fed a
more acidic diet
10
9
Log10 CFU/ml
8
Faecal contents, static cultures
EHEC
O157:H7
Log10 CFU/ml
Rumen contents
Combination of low pH and high VFA
concentration in the rumen lead to EHEC mortality
With Flora
Without Flora
7
6
5
Initial
inoculum
4
3
2
10
9
8
7
6
5
4
3
2
1
0
t0
t24
Without
Flora
1
0
With Flora
The faecal flora does not exert a barrier effect
The ruminal flora exerts a strong barrier effect The faecal environment promotes EHEC growth
EHEC growth is oxygen-dependent
Chaucheyras Durand et al. 2006, AEM
anaerobiosis
aerobiosis
Physiology of EHEC in the bovine gastro-intestinal tract
To investigate expression
of genes involved in
Metabolic pathways
Stress responses
Virulence factors
In the digestive
content of
ruminants
Transcriptomic profiling using microarrays
Methods
Microarray analysis
EDL933
O2
BIC
M9
M9-Gluc
39°C without shaking
cDNAs
mg / ml
What is the carbon source used by EHEC in
the digestive content?
2
Autochthonous Lactic
acid bacteria
Glucose
other
1.5
lldP
Intestinal mucus layer
1
fucA
fucA
fucA
L-Fuculose1P
L-Fuculose1P
L-Fucose
D-Mannose6P
D-Mannose6P
Mannose
0.5
L-Lactate
galU
galE
L-Lactaldehyde
L-Lactaldehyde
x 2
t=0
t=4h30
Mucus-derived
monosaccharides,
fermentation products of
the resident microbiota,
and compounds released
from
degradation
of
intestinal epithelial cells
and from the diet, are
potential carbon sources
for EHEC in the bovine
gut.
-D-glucose1P
Glycolysis
x 1.8
Glycerol
Epithelial
cells
x 2
Citrate
Dihydroxyacetone
Dihydroxyacetone
P P Malate
Malate
TCA
TCAcycle Isocitrate
Isocitrate
fucA
x 2,9
x 3,6
Acetyl-CoA
mdh
mdh Citrate
x 2
glpD x
glpD
x 4,2
4,2
D-Ribulose1P
sn-Glycerol3P3P
glpF
Pyruvate
Oxaloacetate
Oxaloacetate
Oxaloacetate
D-Galactose
D-Arabinose
lldD x 2,3
Fructose 6P
galK x 1.5
D-Galactose
L-Lactate
lldD
galT
0
aldA
aldA
x x
2,1
2,1
xx2,9
2,9
manA
x 2
glpK
glpK x 5,8
Glycerol
fumA
fumA x 2,5
aspA
Oxo-glutarate
glutarate
toglutarate
Fumarate
sdhC
x 4,6
x 5
Succinyl
-CoA
Succinate
Aspartate
Food
dctA x 5.1
Aspartate
Resident microbiota
Succinate
What is the nitrogen source used by EHEC in
the digestive content?
 Amino acid degradation
 Ethanolamine degradation
S
5
P
9
Q
T
D
M
8.7 9.5 8.1 17.1
N
E
J
9.4 3.6 2.8
G
2
H
2
A
B
2.3 1.8
C
L
K
eut gene cluster
R
2.6 2.2 2.2
Fold increase in cattle gut content / minimal medium
Ethanolamine
EutH
NH3
Rumen: 7.7 nM
Rectum: 17.3 nM
EutBC
Acétaldéhyde
EutG
Ethanol
Ethanol
EutE
Acétyl-CoA
EutD
ATP
Acétyl phosphate
TCA
Bertin et al., 2011, Environ. Microbiol.
Acétate
Acétate
Caecum: 12.3 nM
Small intestine: 2200 nM
(≈ x 250)
What is the nitrogen source used by EHEC in
the digestive content?
Phosphatidylethanolamine (PE)
NH33++
CH22
CH22
O
Ethanolamine
EDL933 uses ethanolamine as a nitrogen
source but not as a carbon source
Bertin et al., 2011, Environ. Microbiol.
The ability to metabolize ethanolamine
confers a growth advantage to EDL933 in the
bovine intestinal content
Only 3.7% of bacterial sequenced genomes possess the eut operon. In
particular, bacteria of the Bacteroidetes and Firmicutes phyla,
representing about 99% of the autochthonous digestive microbiota
species, do not possess the eut operon. The eut operon of commensal
E. coli is poorly expressed.
EDL933 may persist in the digestive tract of ruminants
by taking advantage of nutrients that are not consumed
by commensal E. coli and by the normal microbiota
Ethanolamine utilisation as a nitrogen source represents
an ecological niche that confers a competitive advantage
for EHEC strains to persist and develop in the bovine
digestive content
Bertin et al., 2011, Environ. Microbiol.
Stress responses
Alteration of the cell wall
and cell surface structures
Alteration of cell division
Alteration of cell shape
Stress responses
Multi drug resistance
acrA acrB acrR tolC
RND
ydhC
bcrC
emrD
MFS (major facilitator
superfamily)
yidY
- Solvents, dyes, detergents, antibiotics
(novobiocin, erythromycin, fusidic acid,
bacitracin, sulfathiazole, chloramphenicol,
ethidium bromide, biliary acids),
uncouplers of oxidative phosphorylation, - quorum sensing signals
ybjG, mdaA, yciD (ompW)
Chromium, drugs (adriamycin, tetracyclin,
ampicillin)
www.nature.com
Stress responses
AFI
Acid fitness Island
X 0.2 X 0.7
X 0.7 X 0.3 X 0.2 X 0.2
X 0.1
Acid-Resistance
X 0.1 X 0.6
Most of the genes involved in
acid-resistance are downGadE
regulated in BIC.
Acid-Resistance
Phage induction
A number of BP-933W phage genes, including stx2, are down
–regulated in BIC in comparison to M9 medium.
Expression of most of other prophages harbored by EDL933 is
also down-regulated
Virulence factors
Attaching/effacing lesions
Host cell
Stevens et al., IAI, 2002
Bacterial cell
Locus of enterocyte effacement
1.4
4.9
1.6
4.3
1.9
3.3
9.2
4.7
10.4
4.4
8.1
5.7
2.2
6.4
2.8
1.6
3.6
11.3
3.3
1.5
3.3
9.6
1.6
5.6
3.5
2.3
3
0.4
2.1
2.8
1.3
5.5
1.2
1.6
1.8
6.8
3.2
Virulence factors
Adhesive fimbriae: adhesion to epithelial cells
Up-regulated
• F9 involved in colonisation of the bovine gut at other sites than the
recto-anal junction
•Sfa-like, not yet characterized
Down-regulated
• Lpf
Involvement in gut persistence in adult ruminant not clear
• Curli Involvement in gut persistence not investigated
Involved in biofilm formation, invasion of epithelial cell lines
Virulence factors
Grys et al., IAI, 2005
Up-regulation of the StcE protease that cleaves mucus glycoproteins,
facilitating intimate adherence of EHEC to epithelial cells.
Summary
Mucus
Fermentation metabolites
Sfa-like
Fucose arabinose
Lactate Acetate
mannose galactose
F9
Epithelial cells, diet
Glycerol
Ethanolamine
Lipid A-LPS
Nitrogen
source
Carbon
source
Indole
Quorum
sensing signals
Antimicrobials
StcE
Intimin
Type 3
secretion system
Multi-drug
efflux pumps
Effector proteins
Conclusion
Dramatic changes in gene expression allow EHEC to adapt its
physiology to survive in the highly competitive environment
of the cattle digestive content
Competitive probiotics could be selected to target specifically
one or more of the metabolic or stress pathways promoting
EHEC survival in cattle
Future work
 Screening of a panel of probiotic strains (lactic acid bacteria,
yeasts) for their antagonist properties
• on diverse EHEC serotypes
• in cattle digestive contents
• using semi-continuous fermentors
 Effect of the selected probiotic strains
• on crucial pathways previously identified
•on virulence properties (acid resistance, stx-phage release)
Frédérique CHAUCHEYRAS-DURAND
Aurélie AMEILBONNE
Yolande BERTIN
Jordan MADIC
Jean-Pierre GIRARDEAU
Alexandra DURAND
Annie GARRIVIER
Fahima FAQIR
Josée HAREL
Estelle PUJOS
Bernard LYAN
Christine Rozand