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. Debitusand D. Saulnier
Increaseof the world aquaculture production. 60 millions tonnes in 2010 (fish, shellfish) Million tonnes Aquaculture production Fishery production
Pearl oyster(pinctada margaritifera) 2 nd local industry 80% of the colorpearlproduction in the world Moea perles Pinctada margaritifera Blue shrimp(litopenaeus stylirostris) 79,2 T in 2013 Orbicularbatfish(Plataxorbicularis) Onlyfishaquaculture 13 T in 2015 Litopenaeus stylirostris Platax orbicularis The Dive Inn SnipView
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
Antibiotic Bacteriophages Vaccine Probiotic
Antibiotic Bacteriophages Vaccine Probiotic Bacterialresistance
Antibiotic Bacteriophages Vaccine Probiotic Veryspecific Bacterialresistance
Antibiotic Bacteriophages Vaccine Probiotic Expensive Hard to administrate Usefulonlyon fishand not on shellfish
Antibiotic Bacteriophages Vaccine Probiotic Hard to administrate
Antibiotic Bacteriophages Vaccine Probiotic Quorum sensing inhibition
QS = bacterial cell to cell communication Present in many bacterial species Density dependent Auto inducer Auto inducers Auto inducers Lowconcentration No QS High concentration Quorum sensing Activation
Previous studies focused on different pathogen bacteria Staphylococcusaureus Escherichia coli Vibrio fisheri Vibrio harveyi Few studiesfocusedon aquaculture 1 QS inhibitorfoundfor brineshrimps: Halogenated furanones (Defoirdt et al, 2006) Interspecific effect of auto inducer V.splendidus/ V.aestuarianus (De Decker et al, 2007)
Anetzberger
Anetzberger
Bioluminescence Extracellularproducts such as metalloprotease cystein protease haemolysin TypeIII secretion Siderophore Biofilm formation
Investigation of moleculesproduced by marine invertebratesto inhibit the quorum sensing
Benthicinvertebrateof French POlynesie, MArtiniqueand REunionisland: Interaction and evaluationof the chimiodiversityto a durable use of the biodiversity ANR project Start :2012 Duration: 45 months
186 samples harvestedin the 5 archipelagosof French Polynesia
Investigation on the reactionof V.harveyiin presence of organic extracts from sponges (50µg/well) Combine readingbetweenabsorbance (λ=600nm) and Luminescence Utilisation of V.harveyiBB120 wildtype and mutant strains(freeman and Bassler 1999) VibrioharveyiJAF375 : CaptorAI-1 (-), CaptorAI-2 (-), CaptorCAI-1 (+) VibrioharveyiJMH612 : CaptorAI-1 (+), CaptorAI-2 (-), CaptorCAI-1 (-) VibrioharveyiJMH597 : CaptorAI-1 (-), CaptorAI-2 (+), CaptorCAI-1 (-)
BB120 BB120+E1 BB120 BB120+E1 0,2 200000 Absorbance (OD) 0,1 0 Luminescence (RLU) 150000 100000 50000-0,1 0 100 200 300 400 500 600 700 Time (minutes) Absorbance (λ=600nm) 0 0 100 200 300 400 500 600 700 Time (minutes) Luminescence
BB120 BB120+E1 BB120 BB120+E1 0,2 200000 Absorbance (OD) 0,1 0 Luminescence (RLU) 150000 100000 50000-0,1 0 100 200 300 400 500 600 700 Time (minutes) Absorbance (λ=600nm) 0 0 100 200 300 400 500 600 700 Time (minutes) Luminescence
BB120 BB120+E1 BB120 BB120+E1 0,2 200000 Absorbance (OD) 0,1 0 Luminescence (RLU) 150000 100000 Delay 50000-0,1 0 100 200 300 400 500 600 700 Time (minutes) 0 0 100 200 300 400 500 600 700 Time (minutes) Absorbance (λ=600nm) Luminescence
JAF375 JAF375+E1 200000 Luminescence (RLU) 150000 100000 50000 Delay 0 0 100 200 300 400 500 600 700 Time (minutes) JMH597 JMH597+E1 JMH612 JMH612+E1 200000 200000 Luminescence (RLU) 150000 100000 50000 Delay Luminescence (RLU) 150000 100000 50000 Delay 0 0 100 200 300 400 500 600 700 Time (minutes) 0 0 100 200 300 400 500 600 700 Time (minutes)
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. harveyibb120 (wildtype) and 3 mutants 4 differentconcentrations 50 µg.ml -1 10 µg.ml -1 5 µg.ml -1 1 µg.ml -1 No impact on bacterialgrowthat50 µg.ml -1
BB120 JAF 375 JMH 597 JMH 612 Molecule 1 Molecule 2 Molecule 3
BB120 JAF 375 JMH 597 JMH 612 Isonaamine D
BB120 JAF 375 JMH 597 JMH 612 Isonaamine D
BB120 JAF 375 JMH 597 JMH 612 IsonaamineA
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 differentactivation pathways 50 pure moleculetestedon BB120 18 QSI (28%)
Double kineticabsorbance / luminescence Simple method Quorum sensing inhibition highlighted Impact on bacterial growth Mutant collection Target the QS activation pathway Useful for other bacteria
Studyof the virulence afterqsi contact Metalloprotease Biofilm Look for down regulationof gene LuxRgene(super operonof QS) Luciferase