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Received: 22 July 2016    Accepted: 16 February 2017

DOI: 10.1111/anu.12571

ORIGINAL ARTICLE

Identification and characterization of lactic acid bacteria

isolated from rainbow trout (Oncorhynchus mykiss, Walbaum
1792), with inhibitory activity against Vagococcus salmoninarum
and Lactococcus garvieae

B.I. Didinen1  | E.E. Onuk2 | S. Metin1 | O. Cayli1

1
Egirdir Fisheries Faculty, Suleyman Demirel
University, Isparta, Turkey Abstract
2
Faculty of Veterinary Medicine, Department In this study, a total of 98 lactic acid bacteria isolated from rainbow trout intestines
of Aquatic Animal Diseases, Ondokuz Mayıs
were screened for their probiotic properties. The isolates were tested for their ability
University, Samsun, Turkey
to inhibit growth of Vagococcus salmoninarum and Lactococcus garvieae. Based on in
Correspondence
vitro antagonism, 10 isolates were selected and evaluated pathogenicity in rainbow
Behire Isıl Didinen, Egirdir Fisheries Faculty,
Suleyman Demirel University, Isparta, Turkey. trout. Isolates were further investigated for hydrophobicity, bile salts and acid toler-
Email: behiredidinen@hotmail.com
ance. These isolates were able to survive low pH and high bile concentrations and
Funding information showed good adherence characteristics. Isolates were characterized phenotypically,
Scientific and Technological Research Council
and then, 16S rRNA gene sequence analysis was used for confirmation. Selected
of Turkey (TUBITAK), Grant/Award Number:
Project No: 113 O 617; Suleyman Demirel strains were administered orally at 108 cfu/g feed, and fish were challenged with
University BAP (Scientific Research Projects
V. salmoninarum and L. garvieae. The fish fed with lactic acid bacteria supplemented
Coordination Unit), Grant/Award Number:
SDU-BAP -3740-YL2-13 diets did not improve protection against V. salmoninarum. However, administration of
Lactococcus lactis subsp. lactis M17 2-­2 and Lactobacillus sakei 2-­3 resulted in a signifi-
cant reduction in mortality due to L. garvieae when compared to the control fish. RPS
values were calculated as 80 and 53% in fish fed with L. sakei 2-­3 and L. lactis subsp.
lactis M17 2-­2, respectively. Our results suggest that these strains could provide an
alternative for lactococcosis control in aquaculture.

KEYWORDS
aquaculture, lactic acid bacteria, Lactococcus garvieae, probiotic, rainbow trout, Vagococcus
salmoninarum

1 | INTRODUCTION
Lactococcus garvieae is a Gram-­positive pathogen that causes hae-
morrhagic septicaemia and meningoencephalitis in several species
of fish (Pérez-­Sánchez, Balcázar, Garcia, et al., 2011; Vendrell et al.,
2006). Lactococcosis outbreaks have been regularly observed in rain-
bow trout since 2001 in Turkey (Avcı, Aydogan, Tanrıkul, & Birincioğlu,
2010; Diler, Altun, Adıloglu, Kubilay, & Isıklı, 2002; Tanrikul & Gultepe,
2011). Therefore, L. garvieae is now considered as one of the most
important pathogens in the rainbow trout industry in Turkey (Altun,
Onuk, Ciftci, Buyukekiz, & Duman, 2012). Lactococcosis outbreaks in
aquaculture are treated with antibiotics, but these are often ineffec-
tive, and their indiscriminate use has led to an increase in antibiotic
resistance (Pérez-­Sánchez, Balcázar, Garcia, et al., 2011; Romalde &
Toranzo, 2002; Vendrell et al., 2006).
Vagococcosis has emerged in the European trout industry, affect-
ing subadult and adult rainbow trout, with mortality rates of 20%–
80% (Ghittino et al., 1999; Michel, Nougayrede, Eldar, Sochon, & de
Kinkelin, 1997; Ruiz-­Zarzuela, de Blas, Girones, Ghittino, & Mùzquiz,
2005). The disease has been reported in rainbow trout in Turkey
(Didinen et al., 2011; Tanrikul, Avsever, Onuk, & Didinen, 2014).
Vagococcus salmoninarum isolates were found to be susceptible to

400  |  wileyonlinelibrary.com/journal/anu

© 2017 John Wiley & Sons Ltd Aquaculture Nutrition. 2018;24:400–407.
DIDINEN et al.
several drugs in vitro (Didinen et al., 2011; Michel et al., 1997; Ruiz-­
Zarzuela et al., 2005); therefore, antibiotic treatments of vagococcosis
in the field condition could not be effective. In addition, there is no
commercial vaccine available for control of vagococcosis infections.
Lactic acid bacteria (LAB) such as Lactococcus spp., Pediococcus
spp., Lactobacillus spp. and genus Leuconostoc are often used as pro-
biotics in aquaculture because of their beneficial effects (Merrifield
et al., 2010). However, only a few reports on screening and use of
LAB against lactococcosis in rainbow trout have been documented
(Balcázar et al., 2007, 2008; Pérez-­Sánchez, Balcázar, Garcia, et al.,
2011; Pérez-­Sánchez, Balcázar, Merrifield, et al., 2011; Vendrell et al.,
2008). In addition, there is only one study about screening of LAB
against V. salmoninarum (Balcázar et al., 2007).
The aims of this study were to isolate lactic acid bacteria from
rainbow trout and to investigate their ability to inhibit L. garvieae and
V. salmoninarum, pathogenicity in rainbow trout, pH and bile toler-
ances, adherence characteristics and protective effects in lactococ-
cosis and vagococcosis for potential use as probiotics in aquaculture.
2 |  MATERIALS AND METHODS
2.1 | Sampling and isolation of the lactic acid
bacteria
Two fish farms in the Mediterranean region of Turkey were selected
for sampling. The farms were participating in a regular health monitor-
ing programme conducted by Egirdir Fisheries Faculty. The study was
approved by the Local Ethical Committee of the Suleyman Demirel
University (Protocol No: 21438139-­67). A total of 60 healthy rainbow
trout weighing 250 g were collected. Bacterial isolation procedures
were performed in the laboratories of the farms. The fish were eu-
thanized with clove oil at 50 mg/L and aseptically dissected to col-
lect hindgut samples. Samples of each hindgut were homogenized in
phosphate-­buffered saline (PBS). Ten-­fold serial dilutions of intestinal
samples were prepared and spread on de Man, Rogosa and Sharpe
(MRS; Merck) agar plates and incubated at 25°C for 48 hr. Then, colo-
nies were selected, purified by streaking and re-­streaking onto MRS
broth with 15% (v/v) glycerol and stored at −80°C until used for in-
hibitory activity against a culture of V. salmoninarum and L. garvieae.
2.2 | Assessment of antibacterial activity in a well
diffusion agar assay
Well diffusion agar assay (WDAA) was used to determine the antago-
nistic activity of putative probiotic bacteria against the two patho-
gens. V. salmoninarum and L. garvieae strains used in the study were
previously isolated and identified from diseased rainbow trout dur-
ing the natural outbreaks in the farms in the Mediterranean region of
Turkey. These strains were identified using phenotypic tests, species-­
specific PCR assays end sequence analysis (Altun, Diler, & Adiloglu,
2004; Didinen et al., 2011; Diler et al., 2002). The pathogenic bacte-
ria were cultured in 4 ml MRS Broth at 25°C for 48 hr, and 10 μl of
each culture was mixed into 10 ml of melted MRS Agar (43.5–44°C).
|
      401
After solidifying and drying for 15–20 min, wells were punched (diam-
eter, 3 mm) and 10 μl of a 2-­d-­old putative probiotic culture (approx.
108–109 CFU/ml) grown in MRS Broth at 25°C was added to wells
in triplicates. Plates were incubated at 25°C for 24 hr and observed
for clearing zones around the wells. Putative probiotic strains caus-
ing clearing zones in the WDAA were tested once more in MRS Agar
to ensure that the antagonistic activity was stable after storage and
subculture (Hjelm et al., 2004).
2.3 | Possible harmful effects of the bacteria to fish
Isolates, which demonstrated as antagonistic activity against V. sal-
moninarum and L. garvieae, were evaluated further for the lack of harm
into rainbow trout using a bacterial challenge. For this purpose, pu-
tative probiotic cultures were grown in the MRS Broth at 25°C and
subsequently harvested by centrifugation at 5,000 × g for 15 min at
4°C. The supernatant was poured off and the pellet was re-­suspended
with PBS. The cell counts of suspensions were determined by serial
­dilution and the pour plate technique onto MRS Agar and incubated
for 24–48 hr at 25°C. Duplicate groups of 14 fish (250 g) were injected
intraperitoneally (IP) with 0.1 ml of each candidate probiotic bacte-
ria resulting in doses approx. 108 CFU/fish (Irianto & Austin, 2002).
Control groups were injected with 0.1 ml PBS. Re-­isolation of the bac-
teria was performed from the kidney, liver and spleen onto MRS agar,
from all mortalities from each tank during a period of 60 days.
2.4 | Hydrophobicity
To determine the hydrophobicity of the isolates, congo red (0.03%)
was added after sterilization of MRS agar. Each isolate was spread on
plates by the cross-­streak method and incubated at 25°C for 24 hr.
Red colonies were considered positive (hydrophobic), and white or
colourless colonies were considered negative (non-­hydrophobic)
(Leyva-­Madrigal, Luna-­González, Escobedo-­Bonilla, Fierro-­Coronado,
& Maldonado-­Mendoza, 2011; Sharma, Soni, & Meharchandani, 2006).
2.5 | Tolerance to acidic pH and bile salts
Bacterial suspensions were performed in PBS with 107 CFU/ml. The
bacterial suspension was inoculated in sterile PBS or in sterile PBS con-
taining 2.5%–10% (v/v) fish bile (Balcázar et al., 2008; Nikoskelainen,
Salminen, Bylund, & Ouwehand, 2001). Samples were incubated for
1.5 hr at 25°C. After incubation, samples were serially diluted in sterile
PBS and counts of viable bacteria were determined by plate count
using MRS agar. Tolerance at different pH conditions was determined
using a suspension (107 CFU/ml) in PBS adjusted with a pH 1.0–6.5 by
addition of HCl for 1.5 hr at 25°C (Balcázar et al., 2008; Prasad, Gill,
Smart, & Gopal, 1998).
2.6 | Phenotypic characterization
Isolates, which demonstrated antagonistic activity and were non-­
pathogenic to rainbow trout, were characterized by determining
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402       DIDINEN et al.
colony morphology, cell morphology, motility, Gram staining and the
production of cytochrome oxidase and catalase. Further biochemical
characteristics were determined using API 50 CH and API 20 STREP
tests (bioMe′rieux), according to the manufacturers’ instructions.
2.7 | 16S rRNA gene sequence and phylogenetic
analysis
Sequence analysis was performed by Macrogen 16S rRNA full se-
quencing service (Seoul, Korea). Briefly, 16S rRNA gene fragments of
antagonistic strains were amplified by PCR using 27F/1492R primer,
and the amplicon was purified and sequenced by 518F/800R prim-
ers. The resulting sequences were verified by forward and reverse
comparisons, assembled and edited with using Contig Express in
Vector NTI Advance 11.5 (Invitrogen). The obtained consensus se-
quences were compared with previously published data in GenBank
and aligned with previously characterized sequences of closely related
members of the genus, using ClustalW in Mega 5.0 multiple sequence
alignments (Thompson, Higgins, Gibson, & Clustal, 1994). Brevibacillus
brevis (D78457.1) was used as an outgroup. Phylogenetic relation-
ships of the isolates were estimated using the neighbour-­joining (NJ)
method in Mega 5.0 (Tamura et al., 2011). The NJ analysis was per-
formed using a Kimura two-­parameter correction model (Saitou &
Nei, 1987) and pairwise deletion option for gaps. Confidence in the
NJ trees was determined by analysing 1,000 bootstrap replicates
(Felsenstein, 1985) using the Mega program.
2.8 | Preparation of the feed
The selected putative probiotic bacteria were cultured in MRS Broth
for 24 hr at 25°C and harvested by centrifugation at 5,000 × g for
15 min at 4°C. The supernatant was poured off and bacterial pel-
let added into commercial trout feed with vegetable oil at a dose of
8
10  CFU/g (Irianto & Austin, 2002). The probiotic concentration in
each feed was determined by plate counting on MRS agar. The viabil-
ity of presumptive LAB in the feed was evaluated immediately after
feed preparation. The probiotic levels in each feed are almost stable
for 7 days. According to these findings, the feed with bacteria was
prepared every 7 days and then stored at 4°C.
2.9 | Fish and experimental conditions
The experiments were conducted in the flow-­through system. The
water temperature was maintained at 13°C with a flow rate of
1–1.5 L/min. The fish were acclimated for a period of 14 days be-
fore used in the experiment. The experiment groups were carried
out in duplicates. After the acclimation period, the average weight
of the fish was 250 g and the fish were divided into 12 groups and
their duplicates (each group containing 14 fish) into 600-­L tanks.
Two experimental groups were arranged to take into account the
antagonistic activities of the isolates. Therefore, in experiment I for
V. salmoninarum, including duplicates, 16 treatments were fed diets
supplemented with eight different isolates that were found to be
non-­pathogenic and have antagonistic activity against V. salmoni-
narum in early stages of this study, during the trial period at a dose
of 108 CFU/g. Regarding experiment II for L. garvieae, a total of four
groups, including duplicates, were fed diets supplemented with two
different isolates both of which demonstrate antagonistic activities
against L. garvieae, at similar doses to experiment I during the trial
period. Two control groups (each group containing 14 fish) for each
of the two experimental groups were fed unsupplemented diets dur-
ing the experimental stage. Fish in the experiments were fed daily at
1.5% of their biomass for 21 days.
2.10 | Bacterial challenge
The pathogenicity of V. salmoninarum and L. garvieae strains in rain-
bow trout was confirmed with by i.p. injecting and reisolating patho-
gens from diseased fish before experiments.
After 21 days of probiotic feeding, the experimental infection
was carried out by i.p. injection. Pathogen L. garvieae and V. salmon-
inarum strains were grown for 24 hr at 25°C in Tryptic Soy Agar. After
incubation, the cells were harvested by centrifugation (2,000 × g),
washed with PBS and re-­suspended in the same buffer. The num-
ber of bacteria was standardized at 600 nm absorbance. Before the
experiment, all the fish were anesthetized with clove oil (50 mg/L).
Experimental group I and one of the control groups were injected
­intraperitoneally (IP) with 0.1 ml of V. salmoninarum resulting in a dose
of 1.8 × 108 cfu/fish according to Michel et al. (1997). Experimental
group II and its control were challenged with L. garvieae at a dose
1.2 × 107 cfu/fish according to Kubilay, Altun, Ulukoy, Ekici, and Diler
(2008). Dead fish were collected and mortality recorded for a period
of 2 months. Probiotic efficacy was calculated on the last day of the
trial by Relative per cent survival. V. salmoninarum and L. garvieae
were reisolated from the kidney, liver and spleen of freshly dead fish
after the mortalities.
2.11 | Statistical analysis
Data were assessed by one-­way analysis of variance ANOVA, and
Duncan’s multiple range test were used to determine the signifi-
cant variation (p < .05). All statistics were performed using SPSS for
Windows version 15.0 (SPSS, Chicago, USA).
3 | RESULTS
3.1 | Isolation and identification of antagonistic LAB
strains
A total of 98 bacterial isolates obtained from rainbow trout intestine
were screened for antagonistic activity. Six isolates (1-­1, 1-­3, 1-­4, 1-­5,
3-­3 and 3-­5) showed inhibitory activity for V. salmoninarum and two
isolates (2-­1, 2-­2) displayed inhibitory activity for L. garvieae. Two
isolates, 2-­3 and M17 2-­2, showed inhibitory activity against both
V. salmoninarum and L. garvieae. However, the isolates 2-­1 and 2-­2
were found to be pathogenic for rainbow trout and therefore were
DIDINEN et al. |
      403

discharged in the study. All antagonistic isolates presumptively cor-
responded to LAB based on phenotypic characteristics.
The 16S rRNA gene sequences of isolates 1-­1, 1-­3, 1-­4, 1-­5, 2-­3,
3-­3, 3-­5 and M17 2-­2 have been deposited in GenBank databases
under the accession numbers KJ531389, KJ531390, KJ531391,
KJ531392, KJ531395, KJ531396, KJ531397 and KJ531398, re-
spectively. In phylogenetic analyses based on 16S rRNA gene se-
quences, isolates appeared to belong to the genus Lactococcus (1-­1,
1-­3 1-­4, 1-­5, 3-­3 and M17 2-­2), Pediococcus (3-­5) and Lactobacillus
(2-­3) (Figure 1). The 16S rRNA gene sequence from isolates 1-­1, 1-­5
and 3-­3 showed 100% sequence similarity with Lactococcus lactis
subsp. cremoris (GenBank acc #AB008214, NR_074949, KF879177
and AB181302), isolates 1-­3 and 1-­4 showed 100% similarity with
Lactococcus garvieae (GenBank acc #NR_102968, LK985397 and
AB267904), isolate 2-­3 showed 100% similarity with Lactobacillus
sakei (GenBank acc #KM267629, KU317778 and LC064899), and
isolate M17 2-­2 showed 100% similarity with L. lactis subsp. lactis
(GenBank acc #KC754747 and AB601163), while isolate 3-­5 showed
>99.9% sequence identity to Pediococcus acidilactici (GenBank acc
#KM062019 and FJ905315).
3.2 | Hydrophobicity, pH and bile tolerances
All eight isolates were congo red-­positive, indicating the presence of
hydrophobic structures in their cell walls. All L. lactis subsp. cremoris
strains and L. garvieae 1-­3 were resistant to 2.5%–10% bile concen-
trations, while Lactobacillus sakei 2-­3 was resistant to only 2.5% bile
concentration. However, bacterial counts of L. lactis subsp. lactis M17
2-­2, L. garvieae 1-­4 and P. acidilactici 3-­5 significantly reduced in 2.5%
bile concentration (p < .05) (Table 1). L. garvieae 1-­3 and P. acidilac-
tici 3-­5 remained viable after a 1.5-­hr exposure to pH values within
1.5–6.5 range, but none of isolate could tolerate exposure to pH 1.0.
L. lactis subsp. cremoris 3-­3 and 1-­5 strains showed resistance to pH
2.5. Other strains were resistant to pH 3.5 (Table 2).

1-5 (KJ531392.1)
Lactococcus lactis subsp. cremoris strain CF4 (AB181302.1)
76
3-3 (KJ531396.1)
1-1 (KJ531389.1)
Lactococcus lactis subsp. cremoris strain CCMMB1135 (KF879177.1)
Lactococcus lactis subsp. cremoris ATCC 19257 (AB008214.1)
94
Lactococcus lactis subsp. lactis strain CCMMB1040 (KF879115.1)
100 Lactococcus lactis subsp. cremoris strain SK11 (NR_074949.1)
M17 2-2 (KJ531398.1)
Lactococcus lactis subsp. lactis strain Lor-MGB-YQ-2(1) (KC754747.1)
98
100
Lactococcus lactis strain TSB6 (JX291541.1)
90 Lactococcus lactis subsp. lactis strain Ni185 (AB601163.1)

Lactococcus garvieae strain M14 (LK985397.1)

Lactococcus garvieae ATCC 49156 (NR_102968.1)
100
1-3 (KJ531390.1)

95 1-4 (KJ531391.1)
82 Lactococcus garvieae strain NRIC 0611 (AB267904.1)

94 3-5 (KJ531397.1)
100 Pediococcus acidilactici strain JFP1 (KM062019.1)
Pediococcus acidilactici strain Lact11 (FJ905315.2)
100 Lactobacillus sakei subsp. sakei strain JCM 1157 (LC064899.1)
Lactobacillus sakei strain SC1 (KM267629.1)
100
94 2-3 (KJ531395.1)
100 Lactobacillus sakei strain YY4 (KU317778.1)

Brevibacillus brevis (D78457.1)

0.01

F I G U R E   1   Phylogenetic tree based on 16S rRNA gene sequence comparison, obtained with the neighbour-­joining algorithm, showing
the LAB strain with related taxa. Bootstrap values (expressed as a percentage of 1000 replications) >50% are given at the branching points.
Brevibacillus brevis used as an outgroup. Bar, 0.01 substitutions per nucleotide position
 |
404      

T A B L E   1   Tolerance of LAB strains at different bile concentrations for 1.5 hr at 25°C (log cfu/ml, SD*)

Lactococcus lactis subsp. L. lactis subsp. L. lactis subsp. L. lactis subsp. Lactococcus Pediococcus Lactobacillus
% Bile lactis M17 2-­2 cremoris 1-­1 cremoris 3-­3 cremoris 1-­5 garvieae 1-­3 L. garvieae 1-4 acidilactici 3-5 sakei 2-3

0 7.67 (0.32)C 7.25 (0.22) 7.73 (0.44) 7.47 (0.05) 7.12 (0.07) 7.69 (0.31)D 7.43 (0.15)C 7.43 (0.22)B
2.5 6.66 (0.2)Bb 7.04 (0.33)c 7.31 (0.19)d 7.29 (0.31)d 7.22 (0.26)d 5.55 (0.38)Ca 5.77 (0.33)Ba 7.35 (0.07)Bd
Bc d d d d B Bb
5 6.51 (0.39) 7.20 (0.35) 7.27 (0.54) 7.54 (0.21) 7.34 (0.22) 5.16 (0.48) 5.40 (0.13) 4.71 (0.37)Aa
7.5 6.29 (0.15)ABc 7.21 (0.14)d 7.46 (0.14)d 7.62 (0.06)d 7.44 (0.34)d 4.57 (0.5)ABa 5.33 (0.18)Bb 4.50 (0.4)Aa
Abc cd d d d Aa Bb
10 5.89 (0.07) 7.14 (0.1) 7.86 (0.05) 7.44 (0.2) 7.47 (0.1) 3.78 (0.16) 5.43 (0.1) 4.46 (0.27)Aa

Lowercase superscripts denote a significant difference between the LAB strains at the respective bile concentrations (i.e., differences between columns). Capital superscripts denote a significant difference
­between values for the respective strains at different bile concentrations (i.e., differences within columns).
*Bacterial levels were determined by plate counts on MRS. Data are presented as mean (standard deviations).

T A B L E   2   Tolerance of LAB strains at different pH conditions for 1.5 hr at 25°C (log cfu/ml, SD*)

Lactococcus lactis L. lactis subsp. L. lactis subsp. L. lactis subsp. Lactococcus Pediococcus Lactobacillus
pH subsp. lactis M17 2-­2 cremoris 1-­1 cremoris 3-­3 cremoris 1-­5 garvieae 1-­3 L. garvieae 1-4 acidilactici 3-­5 sakei 2-3

1 ND† ND† ND† ND† ND† ND† ND† ND†

Aa † Ac Ab c † d
1.5 1.92 (0.02) ND 5.6 (0.27) 2.96 (0.09) 5.92 (0.03) ND 6.47 (0.34) ND†
2 3.68 (0.14)Ba 3.69 (0.35)Aa 5.57 (0.12)Ad 6.27 (0.30)Be 6.03 (0.38)de 4.33 (0.29)Ab 6.41 (0.11)e 4.92 (0.12)Ac
Ba Ba Bc Bc c ABa c
2.5 4.19 (0.24) 4.55 (0.35) 6.40 (0.31) 6.66 (0.26) 6.47 (0.39) 4.68 (0.09) 6.36 (0.1) 5.33 (0.19)Ab
3 4.04 (0.38)Ba 4.70 (0.49)Bab 6.31 (0.76)Bc 6.65 (0.26)Bc 6.58 (0.16)c 5.28 (0.66)BCb 6.39 (0.1)c 6.50 (0.1)Bc
Cb Cb Bb Bb b BCa b
3.5 6.50 (0.3) 6.66 (0.09) 6.62 (0.54) 6.56 (0.14) 6.57 (0.33) 5.35 (0.1) 6.57 (0.24) 6.53 (0.38)Bb
6.5 6.63 (0.32)Cb 6.43 (0.15)Cb 6.48 (0.19)Bb 6.63 (0.11)Bb 6.59 (0.31)b 5.99 (0.77)Ca 6.45 (0.07)b 6.36 (0.21)Bb

Lowercase superscripts denote a significant difference between the LAB strains at the respective pH level (i.e., differences between columns). Capital superscripts denote a significant difference between values
for the respective strains at different pH concentrations (i.e., differences within columns).
*Bacterial levels were determined by plate counts on MRS. Data are presented as mean (standard deviations).
†
Not detected.
DIDINEN et al.
DIDINEN et al. |
      405

T A B L E   3   Cumulative per cent mortality

Cumulative per cent mortality from
of treated with LAB strains and control
groups challenged with Lactococcus Groups V. salmoninarum L. garvieae
garvieae and Vagococcus salmoninarum
Control 50 53.57A
Lactococcus lactis subsp. lactis M17 2-­2 35.71 24.99B
L. lactis subsp. cremoris 1-­1 42.85
L. lactis subsp. cremoris 3-­3 42.85
L. lactis subsp. cremoris 1-­5 35.71
L. garvieae 1-­3 42.85
L. garvieae 1-­4 49.99
Pediococcus acidilactici 3-­5 35.71
Lactobacillus sakei 2-­3 42.85 10.71B

Capital superscripts denote a significant difference between values for groups at cumulative per cent
mortality (i.e., differences within columns).

2007, 2008; Kim and Austin, 2008; Rengpipat, Rueangruklikhit, &

3.3 | Effect of LAB strains in disease resistance to
vagococcosis and lactococcosis
Cumulative mortalities after challenging with L. garviae were 10.71,
24.99 and 53.57% in fish fed diets containing L. sakei 2-­3, L. lac-
tis subsp. lactis M17 2-­2 and control group, respectively (Table 3).
Statistical analysis demonstrated that fish fed with diet containing L.
sakei and L. lactis subsp. lactis M17 2-­2 at 21 days had significantly
(p < .05) lower mortalities than the control group. Relative per cent
survival were thus 80 and 53% in fish fed with diet containing L. sakei
2-­3 and L. lactis subsp. lactis M17 2-­2, respectively.
Fish fed diets containing LAB strains showed a cumulative mor-
tality ranging from 35.71% (L. lactis subsp. lactis M17 2-­2) to 49.99%
(L. garvieae 1-­4) after challenging with V. salmoninarum. Mortality was
50% in the control group (Table 3). However, no significant differences
(p > .05) were observed among mortalities in all groups. External ex-
amination and bacteriological analysis of fish that died during the
study revealed the presence of L. garvieae and V. salmoninarum in all
cases.
4 |  DISCUSSION
In the present study, we obtained a pool of bacterial isolates from
rainbow trout and characterized those with inhibitory activity against
L. garvieae and V. salmoninarum. Our study demonstrated that 8
(8.16%) of the screened bacteria (n = 98) isolates were antagonistic
to V. salmoninarum and 4 (4.08%) to L. garvieae. These findings sup-
port the view of other studies claiming that antagonism between en-
dogenous gut microorganisms of fish and bacterial pathogens occurs
in nature and the establishment of a normal or protective microbiota
might constitute a key component of defensive barrier function (Cain
& Swan, 2010; Gómez & Balcázar, 2008; Pérez-­Sánchez, Balcázar,
Garcia, et al., 2011). Indeed, several LAB strains isolated from fish
and aquatic animals display antagonistic activity against fish patho-
genic agents (Joborn, Olsson, Westerdahl, Conway, & Kjelle-­berg,
1997; Nikoskelainen et al., 2001; Ringø et al., 2002; Balcázar et al.,
Piyatiratitivorakul, 2008; Ringø et al., 2010; Pérez-­Sánchez, Balcázar,
Garcia, et al., 2011). The previous studies have shown that antagonis-
tic effects against L. garvieae of L. lactis subsp. cremoris DSM 20069
(Balcázar et al., 2007), L. mesenteroides CLFP 196 and L. plantarum
CLFP (Vendrell et al., 2008) and L. plantarum, L. lactis and L. mesen-
teroides strains (Pérez-­Sánchez, Balcázar, Garcia, et al., 2011) isolated
from the intestines of healthy salmonid fish, Weissella viridescens F1,
F4, F9, F10 and F11 strains from Bahia Blanca Estuary (Sica et al.,
2010), and L. garvieae A7 strain isolated from rainbow trout farm
water (Didinen, Metin, Onuk, Takmaz, & Ersoy, 2014). In addition,
Balcázar et al. (2007) reported inhibitory activity of Lactobacillus cur-
vatus (CLFP150) isolated from the intestine of rainbow trout against
V. salmoninarum. However, to our best of knowledge, the inhibition of
V. salmoninarum by Lactococcus lactis subsp. lactis; Lactococcus lactis
subsp. cremoris; Lactococcus garvieae; Lactobacillus sakei; Pediococcus
acidilactici isolates and the inhibition of L. garvieae by Lactobacillus
sakei has not been described before.
In the present study, antagonistic strains against L. garvieae, L.
lactis subsp. lactis 2-­1 ve Weissella viridescens 2-­2 were found to be
pathogenic for rainbow trout when IP injected. The previous studies
also reported the pathogenicity of Weissella spp. for rainbow trout
(Figueiredo, Costa, Leal, Carvalho-­Castro, & Leite, 2012; Liu, Li, Ji, &
Yang, 2009), as well as Lactococcus lactis for Atlantic salmon and rain-
bow trout (Austin & Austin, 2007).
The ability to adhere to intestinal epithelial cells is thought to be
an important characteristic for potential probiotic strains. Pan, Li, and
Liu (2006) assessed the ability of LAB strains to adhere to intestinal
epithelial cells and concluded that the more hydrophobic strains dis-
played stronger adhesive capability. We found that L. lactis subsp. lac-
tis M17 2-­2, L. lactis subsp. cremoris and L. garvieae strains, L. sakei 2-­3
and P. acidilactici 3-­5 were hydrophobic. Our results are in agreement
with previous studies, which demonstrated that L. sakei, L. lactis, L.
plantarum, L. fermentum and L. mesenteroides isolated from rainbow
trout intestine tended to adhere to intestinal mucosa (Balcázar et al.,
2007, 2008; Pérez-­Sánchez, Balcázar, Garcia, et al., 2011). Tolerance
to bile is also considered important for the probiotic strains to grow
 |
406       DIDINEN et al.
and survive in the fish intestine (Nikoskelainen et al., 2001). In our
study, all L. lactis subsp. cremoris strains and L. garvieae 1-­3 were re-
sistant to 2.5%–10% bile concentrations, while Lactobacillus sakei 2-­3
was resistant to only 2.5% bile concentration. Similarly, Balcázar et al.
(2008) noted L. lactis, L. plantarum and L. fermentum can survive at
2.5%–10% bile for 1.5 hr. Indeed, the concentration of bile in the in-
testine of salmonid fish has been estimated to be ranging between
0.4 and 1.3% (Balcázar et al., 2008). Thus, Pérez-­Sánchez, Balcázar,
Garcia, et al. (2011) used 0.2%–1% bile concentrations in their study
and reported tolerance of L. lactis, L. plantarum ve L. mesenteroides to
1.0% bile. The pH of gastric juice is the main factor that determines
the survival of bacteria that pass from the stomach to the intestine
(Balcázar et al., 2008). In this study, Lactococcus lactis subsp. cremoris
3-­3 and 1-­5, Lactococcus garvieae 1-­3 and P. acidilactici 3-­5 showed
resistance to pH 2.5. Similarly, Balcázar et al. (2008) noted L. lactis,
L. plantarum and L. fermentum can survive at pH 2.5. Pérez-­Sánchez,
Balcázar, Garcia, et al. (2011) also reported that L. lactis, L. plantarum
and L. mesenteroides were tolerant at pH 3.0.
To date, the beneficial effects of probiotic administration against
L. garvieae infection in rainbow trout have been demonstrated by in-
corporation of Aeromonas sobria GC2 (Brunt & Austin, 2005), L. mes-
enteroides CLFP 196, L. plantarum CLFP 238 (Vendrell et al., 2008) and
L. plantarum (Pérez-­Sánchez, Balcázar, Merrifield, et al., 2011). To our
knowledge, however, this is the first report describing the probiotic
effect of endogenous L. lactis subsp. lactis M17/2-­2 and L. sakei 2-­3
against L. garvieae infection in rainbow trout.
As a conclusion, LAB isolates from rainbow trout intestines could
serve as a potential probiotics with ability to inhibit pathogen growth
under in vitro conditions, acid and bile tolerance as well as adhesion
to intestinal mucus. Moreover, L. lactis subsp. lactis M17/2-­2 and L.
sakei 2-­3 strains inhibited growth of L. garvieae and V. salmoninarum.
Although these strains decreased mortality caused from lactococcosis,
no significant effect was observed for vagococcosis. Results suggest
that L. lactis subsp. lactis M17/2-­2 and L. sakei 2-­3 strains can provide
a viable alternative for managing lactococcosis in cultured fish.
ACKNOWLE DG E MEN T
This work has been partly supported by Scientific and Technological
Research Council of Turkey (TUBITAK) under Project No: 113 O 617
and partly supported by Suleyman Demirel University BAP (Scientific
Research Projects Coordination Unit) under Project No: SDU-­BAP
-­3740-­YL2-­13.
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How to cite this article: Didinen BI, Onuk EE, Metin S, Cayli O.
Identification and characterization of lactic acid bacteria
isolated from rainbow trout (Oncorhynchus mykiss, Walbaum
1792), with inhibitory activity against Vagococcus
salmoninarum and Lactococcus garvieae. Aquacult Nutr.
2018;24:400–407. https://doi.org/10.1111/anu.12571