7th Rendez vous de Concarneau October 16th 2015

Transcription

1 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

2 Increaseof the world aquaculture production. 60 millions tonnes in 2010 (fish, shellfish) Million tonnes Aquaculture production Fishery production

3 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

4 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

5 Antibiotic Bacteriophages Vaccine Probiotic

6 Antibiotic Bacteriophages Vaccine Probiotic Bacterialresistance

7 Antibiotic Bacteriophages Vaccine Probiotic Veryspecific Bacterialresistance

8 Antibiotic Bacteriophages Vaccine Probiotic Expensive Hard to administrate Usefulonlyon fishand not on shellfish

9 Antibiotic Bacteriophages Vaccine Probiotic Hard to administrate

10 Antibiotic Bacteriophages Vaccine Probiotic Quorum sensing inhibition

11 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

12 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)

13 Anetzberger

14 Anetzberger

15 Bioluminescence Extracellularproducts such as metalloprotease cystein protease haemolysin TypeIII secretion Siderophore Biofilm formation

16 Investigation of moleculesproduced by marine invertebratesto inhibit the quorum sensing

17 Benthicinvertebrateof French POlynesie, MArtiniqueand REunionisland: Interaction and evaluationof the chimiodiversityto a durable use of the biodiversity ANR project Start :2012 Duration: 45 months

18 186 samples harvestedin the 5 archipelagosof French Polynesia

19 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 (-)

20 BB120 BB120+E1 BB120 BB120+E1 0, Absorbance (OD) 0,1 0 Luminescence (RLU) , Time (minutes) Absorbance (λ=600nm) Time (minutes) Luminescence

21 BB120 BB120+E1 BB120 BB120+E1 0, Absorbance (OD) 0,1 0 Luminescence (RLU) , Time (minutes) Absorbance (λ=600nm) Time (minutes) Luminescence

22 BB120 BB120+E1 BB120 BB120+E1 0, Absorbance (OD) 0,1 0 Luminescence (RLU) Delay , Time (minutes) Time (minutes) Absorbance (λ=600nm) Luminescence

23 JAF375 JAF375+E Luminescence (RLU) Delay Time (minutes) JMH597 JMH597+E1 JMH612 JMH612+E Luminescence (RLU) Delay Luminescence (RLU) Delay Time (minutes) Time (minutes)

24 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%)

25 Sponge P30-SBB6, Leucetta chagosensis (Harvested at Bora Bora-Society)

26 Purification of the extract using HPLC 5 molecules obtained Isonaamine A Isonaamine D Leucettamine D

27 Purification of the extract using HPLC 5 molecules obtained Isonaamine A Isonaamine D Leucettamine D New molecules

28 Isonaamidine A Di-isonaamidine D

29 Isonaamidine A Di-isonaamidine D New molecule

30 Challenge of the 5 molecules Using V. harveyibb120 (wildtype) and 3 mutants 4 differentconcentrations 50 µg.ml µg.ml -1 5 µg.ml -1 1 µg.ml -1 No impact on bacterialgrowthat50 µg.ml -1

31 BB120 JAF 375 JMH 597 JMH 612 Molecule 1 Molecule 2 Molecule 3

32 BB120 JAF 375 JMH 597 JMH 612 Isonaamine D

33 BB120 JAF 375 JMH 597 JMH 612 Isonaamine D

34 BB120 JAF 375 JMH 597 JMH 612 IsonaamineA

35 BB120 JAF 375 JMH 597 JMH 612 Leucettamine D

36 BB120 JAF 375 JMH 597 JMH 612 Molecule 4 Molecule 5

37 BB120 JAF 375 JMH 597 JMH 612 Isonaaminidine A

38 BB120 JAF 375 JMH 597 JMH 612 Isonaaminidine A

39 BB120 JAF 375 JMH 597 JMH 612 Di-isonaaminidine D

40 From Leucetta chagosensis 5 pure molecules = 2 QSI on differentactivation pathways 50 pure moleculetestedon BB QSI (28%)

41 Double kineticabsorbance / luminescence Simple method Quorum sensing inhibition highlighted Impact on bacterial growth Mutant collection Target the QS activation pathway Useful for other bacteria

42 Studyof the virulence afterqsi contact Metalloprotease Biofilm Look for down regulationof gene LuxRgene(super operonof QS) Luciferase

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