Download Characterisation of the bacterial flora of pelagic fish

Characterisation of the bacterial flora of pelagic fish,
fish
with emphasis on Atlantic mackerel
(Scomber scombrus)
Cecilie Smith Svanevik
Bjørn Tore Lunestad
Nordic Pelagic Workshop 2010
Gardermoen 30.08.10
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
Fishing expedition
• Norwegian sea,
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
e e pumped
pu ped onboard
o boa d
were
– 4 fish from the fish tank
12 hours after catching
Sampling
Flowchart
• Blue: traditional
microbiological methods
• Red: molecular DNA
analysis
Conventional microbiological methods
3 days, 20 °C
• dfdf
3 days, 20 °C
3 days,
days 20 °C
Results microbiological methods - cfu
• Fish tank vs
vs. Fish net
Bacterial count
100000000
1000000
100000
397
74400
1474
40500
22
276400
10
1750
8000
100
28200
1000
20
024500
10000
65650
Number of bacteria
10000000
1
Gills
Skin
Gut
Gut
anaerobe
N b off colony
Nuber
l
f
forming
i
units
i (cfu)
( f ) in
i 1 gram tissue
i
– Generally higher
numbers in the fish
tank
– Gills and skin samples
differ most
Fish tank
Fish net
Results microbiological methods – H2S
•
H2S producing
d i
b
bacteria
i
10000
1000
312
250
1950
22650
200
400
10
5250
100
4150
number of bacteria
100000
Fish tank
Fish net
1
Gills
Skin
Gut
Gut
anaerobe
Number of H2S producing bacteria of 1 gram tissue
Fish tank vs
vs. Fish net
– Significant higher
number in all
samples from the
fish tank, except the
anaerobe.
– No anaerobe H2S
producing bacteria of
the fish tank
Results microbiological methods - API®
Fish net
Skin
Gut
Gills
Skin
Gut
P t
Proteus
vulgaris
l
i group
Gills
Species/Group
Fish tank
x
x
x
x
x
x
Providencia alcalifaciens/rustigianii
x
x
Stenotrophomonas maltophilia
Empedobacter brevis
x
Shewanella putrefaciens group
x
x
x
Aeromonas salmonicida ssp.
x
Vibrio vulnificus
x
Moraxella spp
pp
x
x
x
x
Brevundimonas diminuta
x
Vibrio alginolyticus
x
Oligella ureolytica
x
• All species/groups
i /
are gram –
• Red: oxidase –
• Blue: oxidase +
Molecular methods
• DNA extracted from
– fish matrix
– from cultured samples
• Amplified
p
by
y Plolymerase
y
Chain Reaction (PCR)
• Separated by Denaturing
p
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 b
by a sequence
seq ence
library (BLAST)
R
Results
lt molecular
l
l
analysis
l i
Species identified by BLAST (sequence library)
DNA from fish matrix and bacterial culture
Fish net
x
x
x
x
x
x
x
x
x
x
x
x
Gut Anaerrobe
x
Gut Aero
obe
Skin
x
Gills
x
x
x
Gut Anaerrobe
x
x
x
x
x
x
x
obe
Gut Aero
x
x
Skin
Psychrobacter immobilis
Psychrobacter sp.
Oceanisphaera sp. V1
sp. V1‐41
41
Proteus sp.
Proteus vulgaris
Shewanella sp.
Shewanella p
putrefaciens strain ZH30
Vibrio sp.
Photobacterium sp.
Mycobacterium sp.
Vagococcus sp. H2914
Vagococcus carniphilus strain 1843‐02
Mycoplasma sualvi
Thiotrichales bacterium clone EC7 Staphylococcus sciuri/ fleurettii
Synechococcus sp.
Uncultured teleost isolate DGGE gel band GL6‐5 18S ribosomal RNA gene
Gills
Species / Groups
Fish tank
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Gram – (red)
• Phylum
proteobactreia
Gram + (blue)
• Phylum
Firmicutes
• Phylum
Cyanobacteria
Teleost DNA
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
Thank you for listening!
Acknowledgement:
•
•
•
•
•
•
•
•
•
•
Kjersti Borlaug, NIFES and IMR
Elise Midthun, NIFES
Betty Irgens, NIFES
A
Arne
L
Levsen,
NIFES
Eva Mykkeltvedt, NIFES
Tone Halvorsen Galluzzi, NIFES
Hui-Shan
Hui
Shan Tung, NIFES
Leikny Fjellstad, NIFES
Sylvia Frantzen, NIFES
Maria Befring Hovda, NOFIMA
Contact info:
Cecilie Smith Svanevik
• [email protected]
• http://no.linkedin.com/in/svanevik
Bjørn Tore Lunestad
• [email protected]