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MolecularAnalysisofMicrobiotaAlongtheDigestiveTract
ofJuvenileAtlanticSalmon(Salmosalar L.)
P. Navarrete• R. T. Espejo • J. Romero
Ô Springer
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digesta,themicrobiota
analyzedwas a combination ofboth DNA Extraction
and Purification
autochthonous(able to colonizethe epithelialsurfaceor
mucusofthehostgut)andallochthonous bacteria(transient). DNA fromthegastrointestinalcompartments was obtained
Simultaneously,freshwater sampleswere obtaineddi- fromhomogenates by lysisusingsodiumdodecylsulfate
rectlyfromthe water influentcorrespondingto the and incubationat 70°C. The lysateswere subsequently
hatchery'swatersourceand thefeedused at thehatchery extractedwith phenol/chloroform and precipitatedwith
were collected and transported on ice. Samples were ethanolas previouslydescribed[41]. A finalpurification
processedimmediately upon arrival
in thelaboratory. was carriedout usingWizardDNA Clean Up (Promega,
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Ô Springer
also platedand incubatedas describedabove. Colonies The presenceof Pseudomonaswas examinedin fish
werecountedafter10 days,and the colony-forming unit smallerthan30 g. TheTTGE profiles obtainedfrom thesefish
(CFU) per gramof gastrointestinal tractwas calculated. (4 to 23 g) did notdiffer the
from TTGE of 30 g (Fig. lc).
50-200colonieswereselectedforanalysis.
Platescontaining Restrictionenzymeanalysisconfirmed thatbandBl inthese
A randomselectionof 80 coloniesfromeach compartment smaller fish also correspondedto Pseudomonas. This
(stomach,pyloriccaeca,andintestine)[38] was subcultured assessment also revealedthepresenceof an additionalband
andcharacterizedaccording to thecolonymorphology, 16S (bandB7) thatwas closely to
related Stenotrophomonas sp.
rDNA restrictionfragment lengthpolymorphism (RFLP),
and ITS profiles,revealingcoincidenceamongthe three Assessedby 16S rDNA Cloning
Diversity
approaches.Representativeisolatesfromeach RFLP group
wereidentifiedby 16S rDNA partialsequencing. To studythemoleculardiversity of bacteriapresentin the
salmon gut, the 16S rDNA ampliconsof the stomach
StatisticalAnalysis sample showingthe largercomplexityin TTGE were
cloned.Eightycloneswereanalyzedby RFLP usingAlul
Assuming100% efficiency whenthe DNA templatewas and Haelll, resulting in six RFLP groups(Table 1). The
doubledin eachcycle,thePCR efficiency was calculatedas mostabundant groupcorresponded to soybeanchloroplasts
£=10(~slope)-l,whereE was the PCR efficiency. PCR (35% of the clones). AnotherthreeRFLP groups(I- III),
efficiencieswere expressedas means±SE of triplicate representing 30%, 20%, and9% oftheclonescorresponded
standardcurves.PCR efficiencies were analyzedwitha to the genus Pseudomonas. Two other RFLP groups
paired t test, and differenceswere concluded to be (IV-V), together representing 6% of theclonesanalyzed,
significant when/?<0.05. corresponded to Acinetobacter. Phylogeneticanalysis
shownin Fig. 2 illustrates themoleculardiversity among
Pseudomonassequencesand revealedthatPseudomonas
Results sequencesfromRFLP groupsIII and I were similarto
TTGE bandBl and bandB2, respectively.
Composition of BacterialCommunities Assessed
by PCR-TTGE Composition of BacterialCommunities Assessed
by ITS Analysis
The bacterialcomposition of stomach,pyloriccaeca, and
intestinefromtenjuvenile(30 g) salmonwas analyzedby CoincidentwiththeTTGE analysis,ITS profilesfromgut
16S rDNA PCR-TTGE. This approachrevealeda simple compartments were similaramongindividuals, showinga
and similarbacterialcompositionin each gastrointestinal dominanceof a widespreadband (650 bp) in all gastroin-
compartment forall individuals.
Five bandswereobserved testinalcompartments (Fig. 3). However,thepresenceof
in the stomachsamples,and two to threebands were severalweakerbandsin theITS profiles mayindicatemore
observed in the pyloric caeca and intestinalsamples diversity in microbiota composition. Sequenceanalysisof
(Fig. lb). TTGE bands with the same migrationpattern the dominant band using BlastN indicated thattheclosest
showed the same restriction profile(Alul and Haelll) match- with 98% -
identitycorresponded Pseudomonas
to
indicating thattheycorresponded to the same sequence fluorescens(accessionnumber EF5877001)andshowedthe
(data not shown). At least one band with the same sameorganization oftRNAs(tRNAIle andtRNAAla) andnon-
migration patternwas sequencedforidentification
(Table 1). codingregionsas wholegenome-sequenced Pseudomonas.
The main bands (Bl and B2) obtainedfromstomach, To testifthepresenceofPseudomonascorresponded to
pyloric caeca, and intestinesamples corresponded to a particular or a general observation, ITS analysiswas
Pseudomonasspp. Threebands observedin the stomach performed in gutsamplesfromfishcollectedfromthesame
samplescorresponded to 16S rDNA of chloroplasts from hatchery butduringdifferent seasons(Fig. 4). The presence
Glycine max (bands B3 and B4) and chloroplastsor of the dominant 650 bp band and the similarITS-RFLP
mitochondrion fromZea mays (band B5), derivedfrom profilesamongthefishsuggeststhata dominant bacterial
the vegetalmaterialincludedin the pelletedfeed,which populationrelatedto Pseudomonaswas presentin salmon
containedapproximately 10% soybeanmeal and 5% corn regardless of theseason(Fig. 4).
gluten.Also, intestinal samplesshowedthepresenceof a
weakband(bandB6) witha 16S rDNA sequencerelatedto BacterialCounts
Comamonassp. This bandwas detectedin 70% (7/10)of
the fish,showingthatthis species, thoughnot always The averagetotalbacterialdensityyielded1x 107,8x 106,
present,was a commoninhabitant of theintestine. and 5xlO7 bacteria/gof stomach,pyloriccaeca, and
Ô Springer
Table 1 Nearest-match of 16S rDNA sequences obtainedfromTTGE bands, cloning approaches,and bacterialisolates from
identification
salmongutcompartments,waterinfluent,and pelletedfeed,withknownsequences in the RDP II database
£} Springer
Table 1 (continued)
£l Springer
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Originof DominantPseudomonasRetrieved
fromSalmon
DigestiveTracts
Discussion
Ö Springer
Ô Springer
revealedby the TTGE analysisof thewaterinfluent and acknowledgesa scholarshipfromCONICYT-Chile and Dr. Stekel a
fromINTA-Nestlé.Partial supportwas derived froman
salmonfeed,Pseudomonasdo notrepresent thedominant fellowship
INNOVA CORFO grant(05CT6PPT-09) and FONDECYT 1080480.
bacteriain these samples,probablybecause theywere
absentor presentin verysmallproportions (less than1%)
[29]. However,a bacterialisolateidentified as Pseudomo-
nas was retrieved fromthe watersample showing97% References
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£) Springer
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4y Springer