High variability of levels of Aliivibrio

and lactic acid bacteria in the intestinal

microbiota of farmed Atlantic salmon
Salmo salar L.
Flix A.Godoy, Claudio D.Miranda,
Geraldine D.Wittwer, Carlos P.Aranda
& Ral Caldern
Annals of Microbiology
ISSN 1590-4261
Volume 65
Number 4
Ann Microbiol (2015) 65:2343-2353
DOI 10.1007/s13213-015-1076-3

1 23

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Author's personal copy

Ann Microbiol (2015) 65:23432353
DOI 10.1007/s13213-015-1076-3

ORIGINAL ARTICLE

High variability of levels of Aliivibrio and lactic acid bacteria

in the intestinal microbiota of farmed Atlantic
salmon Salmo salar L.
Flix A. Godoy 1 & Claudio D. Miranda 2,3 & Geraldine D. Wittwer 1 &
Carlos P. Aranda 1 & Ral Caldern 4

Received: 10 November 2014 / Accepted: 11 March 2015 / Published online: 16 April 2015
# Springer-Verlag Berlin Heidelberg and the University of Milan 2015

Abstract In the present study, the structure of the intestinal

microbiota of Atlantic salmon (Salmo salar L.) was studied
using culture and culture-independent methods. Three adult
specimens of S. salar were collected from a commercial salmon farm in Chile, and their intestinal microbiota were studied
by partial sequencing of the 16S rRNA gene of pure cultures
as well as of clone libraries. Out of the 74 bacterial isolates,
Pseudomonas was the most predominant genus among cultured microbiota. In clone libraries, 325 clones were obtained
from three adult fish, and a total of 36 operational taxonomic
units (OTUs) were identified. This indicated that lactic acid
bacteria (Weissella, Leuconostoc, and Lactococcus genera)
comprised more than 50 % of identified clones in two fishes.
This was in contrast with the high dominance of a single OTU
(99 sequences) of Aliivibrio sp. related to the pathogenic
Aliivibrio salmonicida species and the absence of lactic acid
bacteria in the third fish, suggesting a condition of an asymptomatic non-healthy carrier. It is clear that molecular identification of 16S rRNA gene libraries obtained from intestinal
content samples is effective in determining the overall

* Flix A. Godoy
felix.godoy@ulagos.cl; fegodo@gmail.com
1

Centro i ~ mar, Universidad De Los Lagos, Camino Chinquihue km

6, Casilla 557, Puerto Montt, Chile

Laboratorio de Patobiologa Acutica, Departamento de Acuicultura,

Universidad Catlica del Norte, Larrondo 1281, Coquimbo, Chile

Centro de Estudios Avanzados en Zonas ridas (CEAZA), Larrondo

1281, Coquimbo, Chile

Escuela de Ciencias Ambientales, Facultad de Recursos Naturales,

Universidad Catlica de Temuco, Temuco, Chile

structure of the intestinal microbiota of farmed Atlantic salmon enabling detection of a minority of taxa not previously
reported as part of the intestinal microbiota of salmonids, including the genera Hydrogenophilus, Propionibacterium,
Cronobacter, Enhydrobacter, Veillonella, Prevotella, and
Atopostipes, as well as to evaluate the health status of farmed
fish when evaluating the dominance of potential pathogenic
species and the incidence of lactic acid bacteria.

Keywords Aquaculture . Intestinal microbiota . Aliivibrio .

Salmon farming . Salmo salar

Introduction
Chile is currently a worldwide leading salmon producer and
the Atlantic salmon Salmo salar L. is one of the main Chilean
salmonid farming products, constituting 182,712 tons from
January to June of 2014, and comprising 64 % of total salmonid exports (SalmonChile 2014). It is well established that the
intestinal tract of reared fish harbors a microbiota that fulfill an
important role in immunity, nutrition, and disease control of
reared fishes (Trust and Sparrow 1974; Ring and Birkbeck
1999; Moffitt and Mobin 2006).
To study the microbiota of the gastrointestinal tract of fishes, the general approach has been the use of conventional
culture methods (Ring et al. 1995). However, it has been
found that these methods present several disadvantages and
usually only detect aerobics and facultative anaerobic bacteria,
but do not detect slow-growing bacteria (Spanggaard et al.
2000; Nayak 2010). Thus, molecular analysis of DNA extracted directly from the sample has rapidly replaced cultivation in
the study of the structure of fish intestinal microbiota.

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2344

Nonetheless, when some phenotypic properties such as enzymatic activities need to be studied in order to understand the
potential role of the microbiota in improving fish nutrition, it
is more appropriate to study the fish intestinal microbiota
composition by culture techniques in combination with
culture-independent methods (Bakke-McKellep et al. 2007;
Kristiansen et al. 2011; Askarian et al. 2012).
The intestinal microbiota composition is known to depend
on dietary factors (Gmez and Balczar 2008; Nayak 2010;
Hartviksen et al. 2014). Navarrete et al. (2013), using microbiological analysis, demonstrated that specific bacterial
groups were correlated with the administered diet, and
Reveco et al. (2014) reported that intestinal microbiota of
Salmo salar is sensitive to dietary changes, observing that
the most dominant species were Lactococcus lactis, Weissella
confusa, and Photobacterium phosphoreum. Otherwise,
Navarrete et al. (2008) suggested that Atlantic salmon favors
Pseudomonas establishment because this species was detected as the dominant component in most of the samples
of juvenile farmed Atlantic salmon. There are other studies
that describe the intestinal microbiota of farmed Atlantic
salmon; however, the majority of these studies are associated with fingerling or juvenile stages (Bakke-McKellep
et al. 2007; Navarrete et al. 2008; Cantas et al. 2011;
Navarrete et al. 2013; Reveco et al. 2014).
It has been reported that fish intestinal microbiota have an
important role in regulating nutrient digestion, immune responses, and intestinal differentiation (Bates et al. 2006;
Kanther and Rawls 2010; Merrifield et al. 2010; Nayak
2010; Ray et al. 2012), so physiological and biochemical characterizations of the intestinal isolates are important in elucidating their functions in the gastrointestinal tract. Several studies reported that freshwater fish reared in warm waters harbor
proteolytic, amylolytic, and cellulolytic bacteria in their digestive tracts (Bairaigi et al. 2002; Ghosh et al. 2002; Saha et al.
2006; Kar et al. 2008), whereas it was reported that an increase
in proteolytic enzymes such as trypsin and chymotrypsin in
salmon induces a better assimilation of proteins, as well as an
increase in the growth and stimulation of immune and endocrine systems (Rungruangsak-Torrissen et al. 2009). Additionally, it has been observed that the different bacterial populations composing the intestinal microbiota represent different
metabolic groups, which can enhance the digestive capacity of
fish (Ring and Olsen 1999). Hence, knowledge of the enzymatic capacities of the gastrointestinal microbiota of farmed
salmon could help regulate the intestinal microbiota enhancing nutrition performance of farmed fishes under intensive
rearing conditions. However, only a few studies on adult salmon are available, and knowledge of the enzymatic properties
of the intestinal microbiota of reared salmon is still scarce.
The main aims of this study were to investigate the composition of intestinal microbiota of adult specimens of farmed
Atlantic salmon, Salmo salar L. by culture and cloning

Ann Microbiol (2015) 65:23432353

methods, and to characterize some of the metabolic and enzymatic capabilities of the isolated strains.

Materials and methods

Sampling
Three apparently healthy adult specimens of Atlantic salmon
(Salmo salar L.) with an approximate weight of 2.5 kg were
collected from three different rearing cages belonging to a
commercial salmon farm located at Punta Quilque, X Region,
Chile (13 C, water temperature; 32 g L-1, salinity). Samples
were packed in sterile bags, placed on ice, immediately
transported to the laboratory, and processed within 2 h of
collection.
Sample processing and cultured bacterial count
Adult salmon were externally washed with sterile deionized
water to reduce potential contamination with skin bacteria and
aseptically eviscerated. Salmon intestines were aseptically removed and placed in sterile Petri dishes and were divided into
proximal intestine (defined as the region between the distal
pyloric caeca and widening of the intestine and the appearance
of transverse luminal folds) and distal intestine (the region
from the widening of the intestine and the appearance of transverse luminal folds to anus). Then, digesta from proximal and
distal intestine were gently squeezed out and the two intestinal
segments were thoroughly rinsed three times using 3 mL of
peptone water in order to collect both adherent and nonadherent bacteria (Ring 1993). Culture counts of heterotrophic bacteria were determined by a spread plate method using
Tryptic soy agar (TSA, Difco Labs). Salmon intestinal contents samples were aseptically weighed, ground, and added to
5 mL of sterile physiological saline (0.85 %) (PS) to obtain a
homogenate as previously described by Miranda and
Zemelman (2001). Appropriate tenfold dilution of the homogenates in PS was prepared and 0.1 mL aliquots were inoculated in triplicate onto agar plates. All plates were incubated at
20 C for 510 days and the bacterial numbers per g of
sample were calculated as described in Miranda and
Rojas (2007). A group of representative colonies from
each sample was selected for purity.
Bacterial isolation
Seventy-four isolates were recovered as a representative sample of the intestinal cultured bacterial community of farmed
salmon. From these, 26 strains were recovered from specimen
1 (17 and nine strains from the proximal and distal intestine,
respectively), 25 strains from specimen 2 (14 and 11 strains
from the proximal and distal intestine, respectively) and 23

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Ann Microbiol (2015) 65:23432353

strains from specimen 3 (nine and 14 strains from the proximal

and distal intestine, respectively). The strains were randomly
selected from plates with TSA medium and purified three
times in TSA medium and stored at 85 C in Tryptic soy
broth (Difco Labs) supplemented with 50 % glycerol (2:1)
until use (Gherna 1994).
Bacterial identification by 16S rRNA sequence analysis
Isolates were identified by bacterial 16S rRNA gene sequence
analysis. For amplification of the 16S rRNA genes, crude
DNA extracts were obtained from pure bacterial isolates using
the Wizard genomic purification kit (Promega, Madison, WI,
USA). The 16S rRNA gene was amplified by the polymerase
chain reaction (PCR) using primers 27F 5-AGAGTTTGAT
CMTGGCTCAG-3, 1492R 5-TACGGYTACCTTGTTA
CGACTT-3, and 907R 5-CCGTCAATTCMTTTGAGTT
T-3 (Lane 1991). PCR products were purified using the Wizard SV Gel kit and PCR Clean-up System (Promega) and
sequencing of amplicons was performed by Macrogen (Seoul,
Korea). Identification of partial sequences was performed
using the NCBI BLAST (http://www.ncbi.nlm.nih.gov/), and
strains were considered to belong to the same genus when
similarities in their sequences were 97 % (Rossell-Mora
and Amman 2001). The partial 16S rRNA gene sequences
were submitted to the Genbank database and assigned
accession numbers JF743668 to JF767415.
Phenotypic characterization of isolated strains
The phenotypic tests of Gram's stain, cell morphology, and
oxidase production were determined according to the procedures described in Barrow and Feltham (1993). Additional
phenotypic characteristics were determined by using the API
20E system (bioMrieux, Marcy-lEtoile, France), and strains
were inoculated according to the manufacturers instructions.
API 20E strips were incubated at 20 C for 48 h.
Enzymatic activity of isolated strains
Enzyme production by the salmon intestinal strains were determined utilizing the API ZYM system (bioMrieux), according to the manufacturers guidelines. Briefly, isolated colonies
were cultured overnight in Tryptic soy broth, centrifuged at
5000 g at 4 C and resuspended in sterile 0.8 % (w/v) NaCl
solution to obtain a turbidity of 6 McFarland (1.51.8
109 CFU mL1). This suspension (65 L) was added to each
capsule, and the test strips were incubated for 8 h at 20 C.
Following incubation, one drop of ZYM A (API; tris-hydroxymethyl-aminomethane, hydrochloric acid, sodium
lauryl sulphate, H2O) and one drop of ZYM B (API; fast
blue BB, 2-methoxyethanol) were added to each capsule and

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the color allowed to develop for 5 min and test strips were
read. All assays were performed twice.
16S rRNA gene library construction and sequencing
From a sample of 200 L of the homogenate of the complete
intestinal content containing the proximal and distal intestine
(1:1), bacterial DNA was extracted using the QIAMP DNA
Stool kit (QIAGEN), according to the manufacturers guidelines. Bacterial DNA was verified by the amplification of a
fragment of rRNA 16S gene using the 27 F and 907R universal primers, as was previously described and visualized with
1 % agarose gels. PCR products were purified from agarose
gels with the Wizard SV Gel kit and PCR Clean-up System
(Promega) and cloned using the TOPO TA vector according to
the procedures indicated by Invitrogen. Cultures of
Escherichia coli JM 107 strain were made competent using
the Transform Aid Bacterial Transformation Kit (Fermentas),
following the manufacturers guidelines. Each clone was picked and cultured in LB broth with ampicillin for 16 h. To isolate
plasmidic DNA with the insert, 100 L of liquid culture was
centrifuged at 6000g for 30 min, the medium was discarded,
and the pellet was resuspended in 100 L of sterile water,
incubated at 95 C for 30 min to produce cellular lysis, and
then centrifuged at 6000g for 30 min. Finally, 5 L of the
lysate was amplified to detect the occurrence of the insert,
using the M13F and M13 R primers. PCR products were
verified in 1 % agarose gels, purified and sequenced by
Macrogen (Seoul, Korea).
Sequence analysis
Partial sequences for chimeras using the Chimera Check program from RPD (Ribosomal Database Project) (http://
fungene.cme.msu.edu/FunGenePipeline/chimera_check/
form.spr) were analyzed. Clone sequences in this study were
aligned using the INFERNAL aligner from RDP, secondarystructure aware aligner (Nawrocki and Eddy 2007). Sequences with similarities over 97.0 % were defined as one
phylotype, i.e., one operational taxonomic unit (OTU). The
taxonomic affiliation of the aligned sequences was performed
with Bayesian rRNA Classifier software from the RDP database, using a confidence threshold of 80 % (Wang et al. 2007).
For phylogenetic tree construction, sequences of clones from
fish 2 classified as Aliivibrio sp. were clustered at 97 % sequence identity into OTUs, and aligned with 16S rRNA sequences of the type strains of all species of the genus Aliivibrio
deposited in Genbank (NCBI) using Muscle in the MEGA 6
software (Tamura et al. 2013). Phylogenetic tree was constructed by the Maximum Likelihood method based on the
Tamura-Nei model with 1,000 resampling bootstrap analyses
using MEGA 6 software (Tamura et al. 2013). The partial 16S
rRNA gene sequences obtained have been deposited in

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Ann Microbiol (2015) 65:23432353

GenBank and assigned the accession numbers HQ897283HQ897612.

incidence of gram-positive organisms, belonging to the

Staphylococcus and Bacillus genera (Table 2).

Diversity indices

Enzymatic and metabolic properties of cultured intestinal

microbiota

Biodiversity indices were estimated from clone sequences and

isolated strains. Simpson and Shannon indices were calculated
using software EstimateS 9.1 (http://viceroy.eeb.uconn.edu/
estimates/).

Results
Total cultured bacteria
In general, intestinal samples of the studied salmon exhibited
similar cultured bacterial levels, with bacterial counts on TSA
decreasing from the foregut to the hindgut. Bacterial counts
from proximal portions were always 1 log higher than those of
the distal portions of fish intestine. Proximal portions of fish
intestines were >105 CFU g1, whereas samples from distal
portions of the intestines from all fishes ranged from 103 to
104 CFU g1, as indicated in Table 1.
Diversity of cultured intestinal microbiota
Using culture methods, 74 strains were isolated from intestinal
samples of farmed Atlantic salmon, S. salar. Then the representative isolates were identified based on 16S rRNA gene
sequencing including Proteobacteria, Actinobacteria, and
Firmicutes phyla. Most of the isolated cultured bacteria
belonged to the Proteobacteria Phylum, with a high number
of representatives of the -proteobacteria group. Among these, Pseudomonas (31 strains), Acinetobacter (11 strains), and
Psychrobacter (seven strains) were identified as the most important genera among the cultured intestinal microbiota; they
were being detected in all the sampled fishes (Table 2). No
significant differences among the three sampled fishes as well
as between bacterial strains isolated from proximal and distal
portions of the intestine were detected, with a noticeable low
Table 1 Heterotrophic plate counts (MeanSD of 3 replicates) from
intestinal content samples of farmed salmon Salmo salar L
Sample

Intestine section

Cultured countSD (CFU g1)

Fish 1

Proximal
Distal
Proximal
Distal
Proximal
Distal

5.23105 5.77103
3.07104 3.46103
2.27105 2.52104
7.67103 1.53103
3.07105 7.50104
1.77104 2.08103

Fish 2
Fish 3

No notable differences in the enzymatic profiles obtained by

the API ZYM tests were observed among the intestinal microbiota strains from the sampled fishes. A high incidence of
strains exhibiting the ability to produce the alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase,
valine arylamidase, acid phosphatase, and naphthol-AS-BIphosphohydrolase enzymes (Table 3) was observed. On the
other hand, the production of the -glucoronidase, Nacetyl--glucosaminidase, -mannosidase, and -fucosidase
enzymes was rare (Table 3). When salmon intestinal strains
were analyzed for their capacity to assimilate various substrates, no notable differences among fish samples as well as
between strains isolated from different intestinal portions were
detected. A high incidence of assimilation of citrate, malate,
glucose, and mannose, contrasting with a lower assimilation
of phenyl-acetate, adipate, and maltose was observed
(Table 4).
Diversity of intestinal microbiota by molecular cloning
When intestinal microbiota were studied by using 16S rRNA
cloning, 325 clones were selected and analyzed, mainly from
fishes 1 and 2 (118 and 143 sequences, respectively). An
important degree of variability in the taxonomic diversity of
clones obtained from intestinal samples of sampled fishes was
detected (Fig. 1). A significant dominance of Weissella
(48.3 %) and Leuconostoc (22.0 %) genera was observed
among the intestinal clones from fish 1, whereas Aliivibrio
(81.1 %) was the most dominant genus among the intestinal
clones from fish 2. However, no dominance was detected
among the intestinal clones from fish 3, though Weissella
(25.0 %), Aliivibrio (15.6 %), Leuconostoc (12.5 %),
Acinetobacter (10.9 %), and Lactococcus (10.9 %) were the
more frequent genera (Fig. 1). Using cloning methodologies
to identify the salmon intestinal microbiota, an important incidence of lactic acid bacteria in fish 1 (78.0 %) and 3 (50.0 %)
was observed. It was comprised of the Weissella,
Leuconostoc, Lactococcus, and Enterococcus genera, whereas
only 0.7 % of the intestinal microbiota of fish 2 corresponded
to lactic acid bacteria (Fig. 1).
When the number of genera per sample was considered a
measure of richness, it was observed that the not cultured
microbiota from fishes 1 and 3 exhibited remarkable richness
values greater than those of the cultured intestinal microbiota,
whereas fish 2 showed the opposite due to the high incidence
of representatives of Aliivibrio genus.

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Table 2

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Identification of cultured intestinal microbiota of farmed salmon Salmo salar

Phylum and/or Class

Genus similarity (%)

Similarity (%)

Number of strains
Fish 1

-Proteobacteria

Agrobacterium

91.399.4

Fish 2

P (n=17)

D (n=9)

P (n=14)

Fish 3
D (n=11)

P (n=9)

-Proteobacteria

Brevundimonas

100.0

-Proteobacteria
-Proteobacteria

Acinetobacter
Lelliottia

98.8100.0
100.0

3
1

-Proteobacteria
-Proteobacteria

Luteimonas
Pseudomonas

98.298.3
97.5100.0

-Proteobacteria

Psychrobacter

98.7100.0

-Proteobacteria
Actinobacteria

Stenotrophomonas
Brachybacterium

99.0100.0
99.6

Actinobacteria
Actinobacteria

Kocuria
Microbacterium

99.899.9
97.4

Actinobacteria

Rhodococcus

100.0

Firmicutes/Bacilli
Firmicutes/Bacilli

Bacillus
Staphylococcus

94.6100.0
98.1100.0

1
2

D (n=14)
1

1
1

1
1

1
5

2
1

1
1
1

P Proximal section of intestine, D Distal section of intestine

Table 3 Enzymatic activities of

intestinal microbiota of farmed
salmon determined using the API
ZYM system (bioMrieux)

Activity

Percentage of enzymatic activity

Fish 1

Alkaline phosphatase
Esterase (C4)
Esterase lipase (C8)
Lipase (C14)
Leucine arylamidase
Valine arylamidase
Cystine arylamidase
Trypsin
-Chymotrypsin
Acid Phosphatase
Naphthol-AS-BIphosphohydrolase
-Galactosidase
-Galactosidase
-Glucoronidase
-Glucosidase
-Glucosidase
N-Acetyl--glucosaminidase
-Mannosidase
-Fucosidase
Gelatinase*

Fish 2

Fish 3

P
(n=17)

D
n=11)

P
(n=10)

D
(n=13)

P
(n=12)

D
(n=12)

94.1

100.0

100.0

92.3

100.0

91.7

64.7
88.2
47.1
94.1
82.4
17.6
64.7
29.4
100.0

54.5
81.8
18.2
100.0
45.4
18.2
81.8
18.2
100.0

60.0
90.0
20.0
100.0
40.0
10.0
60.0
20.0
100.0

76.9
92.3
23.1
100.0
76.9
30.8
76.9
7.7
100.0

91.7
83.3
16.7
100.0
66.7
16.7
50.0
33.3
100.0

83.3
66.7
0.0
100.0
58.3
8.3
16.7
0.0
91.7

94.1

100.0

80.0

100.0

91.7

91.7

11.8
23.5
5.9
29.4
41.2
17.6
0.0
0.0
35.3

9.1
18.2
0.0
27.3
54.5
0.0
0.0
0.0
45.4

10.0
20.0
0.0
30.0
30.0
0.0
0.0
0.0
100.0

7.7
30.8
0.0
38.5
46.2
7.7
15.4
7.7
69.2

8.3
16.7
0.0
33.3
25.0
0.0
0.0
0.0
58.3

8.3
8.3
8.3
16.7
8.3
0.0
0.0
0.0
41.7

P Proximal section of intestine, D Distal section of intestine, * Determined using the API 20NE system

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2348
Table 4 Metabolic activities of
intestinal microbiota of farmed
salmon determined using the API
20NE system (bioMrieux)

Ann Microbiol (2015) 65:23432353

Substrate assimilated

Percentage of metabolic activity

Fish 1

Fish 2

Fish 3

P (n=17)

D (n=9)

P (n=12)

D (n=14)

P (n=10)

D (n=11)

Glucose
Arabinose

76.5
64.7

100.0
88.9

91.7
83.3

92.9
85.7

90.0
70.0

63.6
63.6

Mannose

70.6

88.9

83.3

92.9

90.0

81.8

Mannitol
N-acetyl glucosamine

52.9
70.6

88.9
77.8

83.3
83.3

92.9
85.7

80.0
60.0

45.4
45.4

Maltose
Gluconate

29.4
64.7

11.1
88.9

41.7
91.7

35.7
85.7

30.0
80.0

45.4
63.6

Caprate

94.1

77.8

75.0

85.7

60.0

72.7

Adipate
Malate

52.9
94.1

55.6
100.0

25.0
100.0

50.0
92.8

10.0
90.0

9.1
72.7

Citrate
Phenyl-acetate

82.4
23.5

100.0
22.2

100.0
0.0

85.7
0.0

90.0
10.0

81.8
18.2

P Proximal section of intestine, D Distal section of intestine

A total of 113 clones classified as Aliivibrio sp. and clustered

into 14 OTUs were analyzed to provide phylogenetic information. Phylogenetic analysis shows a dominant OTU (represented by clone NG1) containing 99 sequences (87.6 % of all
sequences), which is closely related to pathogenic species
Aliivibrio salmonicida and Aliivibrio logei (Fig. 2).
When diversity indices of the salmon intestinal clones were
estimated, fish 3 presented the highest diversity, with Simpson
and Shannon-Wiener diversity indices of 0.86 and 2.22, respectively, contrasting with the lowest diversity of intestinal
microbiota of fish 2 with Simpson and Shannon-Wiener diversity indices of 0.32 and 0.68, respectively (Table 5). Otherwise, no important differences in the number of genera were
observed between the fishes 1 and 3 (16 and 14, respectively),

Discussion
Most of the currently available information on the intestinal
microbiota of Atlantic salmon Salmo salar refers to the early
stages of growth, mainly juveniles (Navarrete et al. 2008,

100%

Relative abundance

Fig. 1 Relative abundance of

bacterial genera in 16S rRNA
gene clone libraries constructed
from DNA obtained from
intestinal microbiota of farmed
Atlantic salmon (Salmo salar L.).
Genus-level classification was
based on the Classifier tool of the
Ribosomal Database Project
(http://rdp.cme.msu.edu/
classifier/classifier.jsp)

whereas only six genera were detected among the intestinal

microbiota of fish 3 (Table 5). When diversity indices of
clones from intestinal samples were compared to those of
the cultured bacteria, the not cultured bacterial diversity indices were slightly higher than the cultured ones of fishes 1 and
3, but in fish 2 diversity indices of cultured bacteria were
double the diversity indices of intestinal clones (Table 5).

90%

Acinetobacter

Aeromonas

80%

Aliivibrio

Atopostipes

Bacillus

Citrobacter

Cronobacter

Enhydrobacter

60%

Enterobacter

Enterococcus

50%

Enterovibrio

Hydrogenophilus

Lactococcus

Leuconostoc

Photobacterium

Prevotella

Propionibacterium

Pseudomonas

20%

Stenotrophomonas

Streptococcus

10%

Unclassified

Veillonella

Vibrio

Weissella

70%

40%
30%

0%
Fish 1

Fish 2

Fish 3

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Ann Microbiol (2015) 65:23432353

2349

Fig. 2 Phylogenetic tree showing

the relationships between 16S
rRNA sequences of classified
OTUs as Aliivibrio according to
RDP from fish 2 and 16S rRNA
sequences of the type strains of all
species of the genus Aliivibrio
deposited in Genbank (NCBI). A
bootstrap analysis was performed
with 1,000 repetitions. Sequences
of clones are represented by open
circles and sequences
representatives of the genus
Aliivibrio are represented by filled
circles. Numbers in parentheses
indicate the number of sequences
per OTUs

2009; Cantas et al. 2011), but knowledge of intestinal microbiota of adults of Atlantic salmon is still scarce. To our knowledge, this is the first study of the intestinal microbiota of adult
S. salar from intensive farming in Chile.
Most of the previous studies analyzing the cultured fraction
of intestinal microbiota of Atlantic salmon using TSA, reported similar levels of cultured bacteria as found in this study
(Yoshimizu et al. 1976; Huber et al. 2004). Other authors
reported lower levels of cultured counts from salmon intestine
samples such as Navarrete et al. (2008) who found mean
values ranging from 5.01102 to 6.31103 CFU g1 of intestinal content in juveniles of reared Atlantic salmon, and Ring
et al. (2014) who found mean values of 4.17102, 1103 and
2.82102 CFU g1 of proximal, midintestine and distal intestine of S. salar, respectively. In other studies, total viable
counts were determined from adherent and digesta from
midintestine (4.261039.77103 CFU g1 and 4.07103
1.62105 CFU g1, respectively) and distal intestine (8.32
1032.57104 CFU g1 and 9.771032.63105 CFU g1,
respectively) of juveniles of S. salar fed with various

Table 5 Diversity of cultured

and not cultured intestinal
microbiota of farmed salmon

experimental diets (Bakke-McKellep et al. 2007), whereas

Hovda et al. (2007) determined bacterial levels in different sections of the Atlantic salmon gut finding 7.94103 CFU g1 in
the foregut, and 5.01103 CFU g1 in the midgut.
In this study, the dominant genera identified among the
cultured intestinal microbiota isolated using TSA medium
were Pseudomonas, Acinetobacter, and Psychrobacter
(41.89, 14.86, and 9.46 %, respectively). This is in agreement
with Navarrete et al. (2008) who reported that microbiota from
untreated Atlantic salmon in Chile were mainly composed of
Pseudomonas, Acinetobacter, Bacillus, Flavobacterium,
Psychrobacter, and Brevundimonas. This also agrees with a
more recent study by Cantas et al. (2011), which reported a
dominance of representatives of the genera Pseudomonas,
Acinetobacter, and Psychrobacter among the gut bacteria of
juvenile Salmo salar, identified by bacterial culturing and 16S
rRNA PCR techniques. In addition, Pseudomonas sp. and
Acinetobacter sp. have previously been reported as constituting an important part of the intestinal microbiota of salmonids
(Cahill 1990; Ring et al. 2005; Romero and Navarrete 2006;

Fish 1

Number of strains or clones

Number of genera or OTUs
Simpson diversity index
Shannon-Wiener index

Fish 2

Fish 3

CM

UM

CM

UM

CM

UM

26
7
0.66
1.42

118
16
0.72
1.77

25
10
0.76
1.87

143
6
0.32
0.68

23
9
0.83
1.96

64
14
0.86
2.22

CM Cultured microbiota, UM Uncultured microbiota using 16S rRNA clone libraries

Author's personal copy

2350

Hovda et al. 2007). Furthermore, recent reports have demonstrated the presence of different Psychrobacter species in the
alimentary tract of Atlantic salmon (Ring et al. 2006a; 2008),
as well as the distal portion of the intestine of Arctic charr
(Ring et al. 2006b).
In this study a high proportion of the isolated bacteria from
the intestinal content of Atlantic salmon exhibited enzymatic
activities, suggesting a potential role in the degradation and
assimilation of nutrients, contributing to the nutrition of reared
salmon, but the low bacterial counts suggest a poor contribution of bacterial enzymes to the degradation of macronutrients.
Furthermore, it is important to note that intestinal transit is
short, and the rearing temperature is much lower than those
used for in vitro enzymatic assays and the API ZYM profiles
are insufficient and other enzymatic assays are required to
evaluate the possible contribution to digestion by gut microbiota. More research is required to understand the potential
function of intestinal microbiota as a source of digestive enzymes in farmed salmon, and the feasibility of its use in enhancing nutrient utilization and growth rate when they are fed
with formulated diets.
As has been noted, when intestinal microbiota were studied
by using culture-dependent methods, most of the cultured organisms belonged to the -subclasses of the proteobacteria
(Pseudomonas sp. and Acinetobacter sp.), in contrast to the
intestinal microbiota revealed by the results of direct cloning
methods, that exhibited a predominance of lactic acid bacteria,
which comprised more than 36 % of the identified clones.
This proportion could have been higher, but for the unexpected high dominance of representatives belonging to the
Aliivibrio genus in fish 2. The fact that lactic acid bacteria
from fish are commonly slow growing, requiring growth conditions on agar-media at low temperatures for up to four weeks
(Ring and Gatesoupe 1998), would explain why lactic acid
bacteria were only detected from 16S rDNA clones libraries,
and not from the intestinal microbiota obtained after cultured
on TSA, given that this medium is not suitable for growth of
this bacterial group.
It is well established that lactic acid bacteria constitute a
part of the native microbiota of fish (Ring 2004; Hovda et al.
2012; Ring et al. 2014). Our results are in agreement with a
recent study, showing the taxonomic affiliation of DGGE
band sequences from the midintestine and distal intestine content of Atlantic salmon.This study is based on known sequences of 16S rRNA genes that indicated a high dominance
of lactic acid bacteria mainly composed of the Weissella and
Lactococcus genera, whereas Photobacterium was the most
representative of -proteobacteria (Reveco et al. 2014). Along
with this, within the lactic acid bacteria there is a high dominance of the genus Weissella from specimens 1 and 3. Various
strains belonging to this genus have previously been proposed
as potential probiotics for various fish species (Cai et al. 1998;
Balczar et al. 2008) However, recent cases of Weissellosis in

Ann Microbiol (2015) 65:23432353

salmonids (Liu et al. 2009; Figueiredo et al. 2012), as well as

the experimental development of disease after its inoculation,
demonstrated the role of the primary pathogen of some strains
identified as Weissella sp., confirming Weissellosis as an
emerging disease in rainbow trout aquaculture (Marancik
et al. 2013; Welch and Good 2013).
When comparing diversity of the intestinal microbiota of
farmed Atlantic salmon obtained by analysis of the 16S rRNA
gene of cultured strains and clone libraries, the diversity of
clone libraries was higher than those from the cultured microbiota, with the exception of fish 2, which exhibited an unexpectedly high dominance of Aliivibrio representatives when
its clone library was identified. Despite the absence of external
and internal symptoms of vibriosis when fish 2 was sampled,
there was a high incidence of Aliivibrio genus in the intestinal
microbiota, suggesting that the sampled fish was under an
initial stage of infection by this strain. On the other hand, the
absence of Aliivibrio in the cultured microbiota is probably
due to the use of TSA medium without addition of NaCl.
Currently, Aliivibrio genus comprises five species: A. fischeri,
A. logei, A. salmonicida, A. wodanis, and A. finisterrensis
(Beaz-Hidalgo et al. 2010). From these species, A. salmonicida
(Egidius et al. 1986), A. wodanis (Lunder et al. 2000), and
A. logei (Benediktsdottir et al. 1998) have been associated
with disease in Atlantic salmon. Furthermore, it has been
demonstrated that A. salmonicida is able to colonize the fish
intestinal tract (Hansen and Olafsen 1999). Further studies
are needed to determine whether high levels of Aliivibrio
spp. as observed in fish 2, are detrimental to the sanitary
status of reared salmon under intensive conditions, because
the increase in the concentration of this genus could be a
response to an infection with a pathogenic Aliivibrio strain
causing an imbalance in the structure of the intestinal
microbiota.
It is important to note that fish 2 not only exhibited a
remarkably higher level of Aliivibrio sp. than that observed in the other two sampled individuals, but also
showed almost an absence of lactic acid bacteria, contrasting with the observations in the other sampled fishes. It
must be noted that the importance of the interaction between lactic acid bacteria and pathogens in the intestines
of salmon species prevents intestinal cellular damage
(Ring et al. 2010).
In a previous study, it was demonstrated that Vibrio
(Aliivibrio) salmonicida can colonize the salmon intestine,
which creates healthy carriers (Bjelland et al. 2012), but this
state only occurs without the bacteria dominating the ubiquitous gut microbiota. In this study, the high predominance of
Aliivibrio in the intestinal microbiota of fish 2 suggests that
fish 2 was an asymptomatic non-healthy carrier, but the other
two sampled individuals were healthy carriers. It is important
to note that all sampled fishes had the same origin and rearing
history. The only difference was that they came from different

Author's personal copy

Ann Microbiol (2015) 65:23432353

cages from adjacent cage blocks belonging to the same

company.
The use of PCR-based identification of cultured organisms
in combination with cloning of 16S rRNA genes, amplified
directly from salmon intestinal samples, are necessary to properly study the structure and some functions of salmon intestinal
microbiota. With this procedure, it must be noted that only the
cultivation-independent analysis could detect a minority taxa
not previously reported as part of the intestinal microbiota of
salmonids, including the Hydrogenophilus, Propionibacterium,
Cronobacter, Enhydrobacter, Veillonella, Prevotella, and
Atopostipes genera, contributing to the knowledge of the microbiota of the digestive tract of farmed Atlantic salmon.
In a previous study, it was found that the microbial intestinal community composition varies significantly in individual
Atlantic cod specimens caught at a single location, and the
authors suggested that such high variation among specimens
is due to a complex combination of factors (Star et al. 2013).
The observed high variability is not unexpected, but further
studies are required to understand these variations.
In conclusion, this study permitted acquisition of knowledge of the structure of intestinal microbiota of S. salar cultured under intensive rearing conditions. There was evidence
of a high variability of levels of Aliivibrio sp. among individuals collected from different cages of the same farm. The high
predominance of a single clone of Aliivibrio sp. related to the
pathogenic A. salmonicida and A. logei species in a sampled
fish suggest its infection by an Aliivibrio strain capable of
avoiding fish defense mechanisms and proliferating in the
intestinal environment previous to the production of noticeable symptoms of disease. In addition, the absence of lactic
acid bacteria in the intestine of this individual is probably
related to its high load of Aliivibrio. It is clear that molecular
identification of 16S rRNA gene libraries obtained from intestinal content samples is effective in determining the overall
structure of the intestinal microbiota of farmed Atlantic salmon enabling evaluation of the health status of farmed fish when
evaluating the dominance of potential pathogenic species as
well as the incidence of lactic acid bacteria.
Acknowledgments The comments and suggestions of one anonymous
reviewer are greatly appreciated as they helped to improve the presentation of this work.

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