7th rendez vous de concarneau october 16th...
TRANSCRIPT
7th Rendez vous de Concarneau
October 16th 2015
K. Magré, T. Mai, F. Tintillier, A. Lucasson, E. Aliotti, C. Moriou,
E. Bonno, S. Petek, A. Al Mourabit, C. Debitus and D. Saulnier
� Increase of the world aquaculture production.
� 60 millions tonnes in 2010 (fish, shellfish)
Fishery productionAquaculture production
Million tonnes
� Pearl oyster (Pinctada margaritifera)
� 2nd local industry
�80% of the color pearl production in the world
� Blue shrimp (Litopenaeus stylirostris)
�79,2 T in 2013
� Orbicular batfish (Platax orbicularis )
�Only fish aquaculture
�13 T in 2015Platax orbicularis ©
Th
e D
ive
Inn
Pinctada margaritifera
©M
oe
ap
erl
es
Litopenaeus stylirostris ©S
nip
Vie
w
� Vibrio genus (more than 80 species)
� Present in all marine environments
� Planktonic bacteria or biofilm
� Vibrio harveyi and Tenacibaculum maritimum
� Present in French Polynesia
� Causing septicaemia on the cultured Batfish
� Pearl oyster death and nucleus release after grafting
Economic losses
ProbioticAntibiotic VaccineBacteriophages
ProbioticAntibiotic VaccineBacteriophages
Bacterial resistance
ProbioticAntibiotic VaccineBacteriophages
Very specific
Bacterial resistance
ProbioticAntibiotic VaccineBacteriophages
Expensive
Hard to administrate
Useful only on fish and not on shellfish
ProbioticAntibiotic VaccineBacteriophages
Hard to administrate
ProbioticAntibiotic VaccineBacteriophages
Quorum sensing
inhibition
� QS = bacterial cell to cell communication� Present in many bacterial species� Density dependent
Low concentration
No QSHigh concentration
Auto inducer
Auto inducersAuto inducers
Quorum sensing Activation
� Previous studies focused on different pathogenbacteria� Staphylococcus aureus
� Escherichia coli
� Vibrio fisheri
� Vibrio harveyi
� Few studies focused on aquaculture � 1 QS inhibitor found for brine shrimps : Halogenated
furanones (Defoirdt et al, 2006)
� Interspecific effect of auto inducer� V.splendidus / V.aestuarianus (De Decker et al, 2007)
Anetzberger
Anetzberger
•Bioluminescence
•Extracellular products such as
metalloprotease
cystein
protease
haemolysin
•Type III secretion
•Siderophore
•Biofilm formation
� Investigation of molecules produced
by marine invertebrates to inhibit
the quorum sensing
� Benthic invertebrate of French POlynesie,
MArtinique and REunion island : Interaction
and evaluation of the chimiodiversity to a
durable use of the biodiversity
� ANR project
� Start : 2012
� Duration : 45 months
186 samples
harvested in the 5
archipelagos of
French Polynesia
� Investigation on the reaction of V.harveyi in presence of organic extracts from sponges(50µg/well)
� Combine reading between Absorbance (λ=600nm)
and Luminescence
� Utilisation of V.harveyi BB120 wild type and mutant strains (Freeman and Bassler 1999)
�� Vibrio harveyi JAF375 : Captor AI-1 (-), Captor AI-2 (-), Captor CAI-1 (+)� Vibrio harveyi JMH612 : Captor AI-1 (+), Captor AI-2 (-), Captor CAI-1 (-)� Vibrio harveyi JMH597 : Captor AI-1 (-), Captor AI-2 (+), Captor CAI-1 (-)
-0,1
0
0,1
0,2
0 100 200 300 400 500 600 700
Ab
sorb
an
ce (
OD
)
Time (minutes)
BB120 BB120+E1
0
50000
100000
150000
200000
0 100 200 300 400 500 600 700L
um
ine
sce
nce
(RL
U)
Time (minutes)
BB120 BB120+E1
Absorbance(λ=600nm)
Luminescence
-0,1
0
0,1
0,2
0 100 200 300 400 500 600 700
Ab
sorb
an
ce (
OD
)
Time (minutes)
BB120 BB120+E1
0
50000
100000
150000
200000
0 100 200 300 400 500 600 700L
um
ine
sce
nce
(RL
U)
Time (minutes)
BB120 BB120+E1
LuminescenceAbsorbance(λ=600nm)
-0,1
0
0,1
0,2
0 100 200 300 400 500 600 700
Ab
sorb
an
ce (
OD
)
Time (minutes)
BB120 BB120+E1
0
50000
100000
150000
200000
0 100 200 300 400 500 600 700L
um
ine
sce
nce
(RL
U)
Time (minutes)
BB120 BB120+E1
Luminescence
Delay
Absorbance(λ=600nm)
0
50000
100000
150000
200000
0 100 200 300 400 500 600 700
Lu
min
esc
en
ce (R
LU
)
Time (minutes)
JAF375 JAF375+E1
0
50000
100000
150000
200000
0 100 200 300 400 500 600 700
Lu
min
esc
en
ce (R
LU
)
Time (minutes)
JMH597 JMH597+E1
0
50000
100000
150000
200000
0 100 200 300 400 500 600 700
Lu
min
esc
en
ce (R
LU
)
Time (minutes)
JMH612 JMH612+E1
Delay
Delay
Delay
BB120 (Wild type) strain Organic extracts
N=186
QSI activity 76 (41%)
Antibiotic activity 18 (10%)
No impact 82 (44%)
Data not clear 10 (5%)
Mutant strain JAF375 JMH597 JMH612
Organic extract N=50
QSI activity 38 (76%) 45 (90%) 31 (62%)
Antibiotic activity 6 (12%) 3 (6%) 9 (18%)
No impact 2 (4%) 2 (4%) 9 (18%)
Data not clear 4 (8%) 0 2 (4%)
Sponge P30-SBB6, Leucetta chagosensis
(Harvested at Bora Bora-Society)
� Purification of the extract using HPLC
� 5 molecules obtained
Isonaamine A
Isonaamine D Leucettamine D
� Purification of the extract using HPLC
� 5 molecules obtained
Isonaamine A
Isonaamine D Leucettamine D
New molecules
Isonaamidine A Di-isonaamidine D
Isonaamidine A Di-isonaamidine D
New molecule
� Challenge of the 5 molecules� Using V. harveyi BB120 (wild type) and 3
mutants � 4 different concentrations
� 50 µg.ml-1
� 10 µg.ml-1
� 5 µg.ml-1
� 1 µg.ml-1
� No impact on bacterial growth at 50 µg.ml-1
BB120 JAF 375 JMH 597 JMH 612
Molecule 1
Molecule 2
Molecule 3
Isonaamine D
BB120 JAF 375 JMH 597 JMH 612
BB120 JAF 375 JMH 597 JMH 612
Isonaamine D
Isonaamine A
BB120 JAF 375 JMH 597 JMH 612
BB120 JAF 375 JMH 597 JMH 612
Leucettamine D
BB120 JAF 375 JMH 597 JMH 612
Molecule 4
Molecule 5
BB120 JAF 375 JMH 597 JMH 612
Isonaaminidine A
BB120 JAF 375 JMH 597 JMH 612
Isonaaminidine A
BB120 JAF 375 JMH 597 JMH 612
Di-isonaaminidine D
� From Leucetta chagosensis� 5 pure molecules = 2 QSI on different activation
pathways
� 50 pure molecule tested on BB120� 18 QSI (28%)
� Double kinetic absorbance / luminescence
� Simple method
� Quorum sensing inhibition highlighted
� Impact on bacterial growth
� Mutant collection
� Target the QS activation pathway
� Useful for other bacteria
� Study of the virulence after QSI contact
� Metalloprotease
� Biofilm
� Look for down regulation of gene
� LuxR gene (super operon of QS)
� Luciferase