with emphasis on Atlantic mackerel (Scomber scombrus) Cecilie Smith Svanevik Bjørn Tore Lunestad Nordic Pelagic Workshop 2010 Gardermoen

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1 Characterisation of the bacterial flora of pelagic fish, with emphasis on Atlantic mackerel (Scomber scombrus) Cecilie Smith Svanevik Bjørn Tore Lunestad Nordic Pelagic Workshop 2010 Gardermoen

2 Master thesis Characterising the bacterial flora of pelagic fish Compare traditional microbiological methods with molecular methods Compare three types of tissue; gills, skin and gut. Compare samples collected from the fish net to those collected from the fish tank

3 Fishing epedition Norwegian sea, October 2009 Total catch of 180 metric tonnes mackerel I collected a total of 8 fish 4 fish from the fish net before the fish were e pumped ped onboard 4 fish from the fish tank 12 hours after catching

4 Sampling

5 Flowchart Blue: traditional microbiological methods Red: molecular DNA analysis

6 Conventional microbiological methods 3 days, 20 C dfdf 3 days, 20 C 3 days, 20 C

7 Results microbiological methods - cfu of bacteria Number Bacterial count Fish tank vs. Fish net Generally higher numbers in the fish tank Gills and skin samples differ most Fish tank Fish net 1 Gills Skin Gut Gut anaerobe Nuber of colony forming units (cfu) in 1 gram tissue

8 Results microbiological methods H 2 S of bacteria number H 2 S producing bacteria Fish tank Fish net Fish tank vs. Fish net Significant higher number in all samples from the fish tank, ecept the anaerobe. No anaerobe H 2 S producing bacteria of the fish tank 1 Gills Skin Gut Gut anaerobe Number of H 2 S producing bacteria of 1 gram tissue

9 Results microbiological methods - API Fish net Fish tank Species/Group Gills Skin Gut Gills Skin Gut Proteus vulgaris group All species/groups Providencia alcalifaciens/rustigianii are gram Stenotrophomonas maltophilia Red: oidase Empedobacter brevis Shewanella putrefaciens group Aeromonas salmonicida ssp. Blue: oidase + Vibrio vulnificus Moraella spp Brevundimonas diminuta Vibrio alginolyticus Oligella ureolytica

10 Molecular methods DNA etracted from fish matri from cultured samples Amplified by Plolymerase Chain Reaction (PCR) Separated by Denaturing Gradient Gel Electrophoresis (DGGE) Samples loaded in a polyacrylamide gel with a denaturing gradient from 30 % to 55 % Connected to electric current The gel was run for 18 hours at 70 V Sequenced by a sequence laboratory Identified by a sequence ence library (BLAST)

11 Results molecular l analysis Species identified by BLAST (sequence library) DNA from fish matri and bacterial culture Fish net Fish tank Species / Groups Gills Psychrobacter immobilis Psychrobacter sp. Gram (red) Oceanisphaera sp. V1 41 Phylum Proteus sp. Proteus vulgaris proteobactreia Shewanella sp. Gram + (blue) Shewanella putrefaciens strain ZH30 Phylum Vibrio sp. Firmicutes Photobacterium sp. Mycobacterium sp. Phylum Vagococcus sp. H2914 Cyanobacteria Vagococcus carniphilus strain Mycoplasma sualvi Teleost DNA Thiotrichales bacterium clone EC7 Staphylococcus sciuri/ fleurettii Synechococcus sp. Uncultured teleost isolate DGGE gel band GL6 5 18S ribosomal RNA gene Skin Gut Aero obe Gut Anaer robe Gills Skin Gut Aero obe Gut Anaer robe

12 Thoughts An increased knowledge about the bacterial flora of the fish could result in a better utilisation of harvested resources Higher number of bacteria in the fish tank Handling activity could cause contamination of the fish from gut content Mostly harmless or opportunistic pathogen species Methods Microbiological method (API tests) necessary temperature could not be used designed for clinical isolates Molecular method (PCR DGGE) reliable results discovers species that are not possible to culture

13 Thank you for listening! Acknowledgement: Kjersti Borlaug, NIFES and IMR Elise Midthun, NIFES Betty Irgens, NIFES Arne Levsen, NIFES Eva Mykkeltvedt, NIFES Tone Halvorsen Galluzzi, NIFES Hui-Shan Tung, NIFES Leikny Fjellstad, NIFES Sylvia Frantzen, NIFES Maria Befring Hovda, NOFIMA Contact info: Cecilie Smith Svanevik Bjørn Tore Lunestad