7th rendez vous de concarneau october 16th...

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