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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 aquaculture are treated with antibiotics, but these are often ineffec-
tive, and their indiscriminate use has led to an increase in antibiotic
Lactococcus garvieae is a Gram-­positive pathogen that causes hae- resistance (Pérez-­Sánchez, Balcázar, Garcia, et al., 2011; Romalde &
morrhagic septicaemia and meningoencephalitis in several species Toranzo, 2002; Vendrell et al., 2006).
of fish (Pérez-­Sánchez, Balcázar, Garcia, et al., 2011; Vendrell et al., Vagococcosis has emerged in the European trout industry, affect-
2006). Lactococcosis outbreaks have been regularly observed in rain- ing subadult and adult rainbow trout, with mortality rates of 20%–
bow trout since 2001 in Turkey (Avcı, Aydogan, Tanrıkul, & Birincioğlu, 80% (Ghittino et al., 1999; Michel, Nougayrede, Eldar, Sochon, & de
2010; Diler, Altun, Adıloglu, Kubilay, & Isıklı, 2002; Tanrikul & Gultepe, Kinkelin, 1997; Ruiz-­Zarzuela, de Blas, Girones, Ghittino, & Mùzquiz,
2011). Therefore, L. garvieae is now considered as one of the most 2005). The disease has been reported in rainbow trout in Turkey
important pathogens in the rainbow trout industry in Turkey (Altun, (Didinen et al., 2011; Tanrikul, Avsever, Onuk, & Didinen, 2014).
Onuk, Ciftci, Buyukekiz, & Duman, 2012). Lactococcosis outbreaks in 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. |
      401

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

2.9 | Fish and experimental conditions


3 | RESULTS
The experiments were conducted in the flow-­through system. The
water temperature was maintained at 13°C with a flow rate of
3.1 | Isolation and identification of antagonistic LAB
1–1.5 L/min. The fish were acclimated for a period of 14 days be-
strains
fore used in the experiment. The experiment groups were carried
out in duplicates. After the acclimation period, the average weight A total of 98 bacterial isolates obtained from rainbow trout intestine
of the fish was 250 g and the fish were divided into 12 groups and were screened for antagonistic activity. Six isolates (1-­1, 1-­3, 1-­4, 1-­5,
their duplicates (each group containing 14 fish) into 600-­L tanks. 3-­3 and 3-­5) showed inhibitory activity for V. salmoninarum and two
Two experimental groups were arranged to take into account the isolates (2-­1, 2-­2) displayed inhibitory activity for L. garvieae. Two
antagonistic activities of the isolates. Therefore, in experiment I for isolates, 2-­3 and M17 2-­2, showed inhibitory activity against both
V. salmoninarum, including duplicates, 16 treatments were fed diets V. salmoninarum and L. garvieae. However, the isolates 2-­1 and 2-­2
supplemented with eight different isolates that were found to be were found to be pathogenic for rainbow trout and therefore were
DIDINEN et al. |
      403

discharged in the study. All antagonistic isolates presumptively cor- (GenBank acc #KC754747 and AB601163), while isolate 3-­5 showed
responded to LAB based on phenotypic characteristics. >99.9% sequence identity to Pediococcus acidilactici (GenBank acc
The 16S rRNA gene sequences of isolates 1-­1, 1-­3, 1-­4, 1-­5, 2-­3, #KM062019 and FJ905315).
3-­3, 3-­5 and M17 2-­2 have been deposited in GenBank databases
under the accession numbers KJ531389, KJ531390, KJ531391,
3.2 | Hydrophobicity, pH and bile tolerances
KJ531392, KJ531395, KJ531396, KJ531397 and KJ531398, re-
spectively. In phylogenetic analyses based on 16S rRNA gene se- All eight isolates were congo red-­positive, indicating the presence of
quences, isolates appeared to belong to the genus Lactococcus (1-­1, hydrophobic structures in their cell walls. All L. lactis subsp. cremoris
1-­3 1-­4, 1-­5, 3-­3 and M17 2-­2), Pediococcus (3-­5) and Lactobacillus strains and L. garvieae 1-­3 were resistant to 2.5%–10% bile concen-
(2-­3) (Figure 1). The 16S rRNA gene sequence from isolates 1-­1, 1-­5 trations, while Lactobacillus sakei 2-­3 was resistant to only 2.5% bile
and 3-­3 showed 100% sequence similarity with Lactococcus lactis concentration. However, bacterial counts of L. lactis subsp. lactis M17
subsp. cremoris (GenBank acc #AB008214, NR_074949, KF879177 2-­2, L. garvieae 1-­4 and P. acidilactici 3-­5 significantly reduced in 2.5%
and AB181302), isolates 1-­3 and 1-­4 showed 100% similarity with bile concentration (p < .05) (Table 1). L. garvieae 1-­3 and P. acidilac-
Lactococcus garvieae (GenBank acc #NR_102968, LK985397 and tici 3-­5 remained viable after a 1.5-­hr exposure to pH values within
AB267904), isolate 2-­3 showed 100% similarity with Lactobacillus 1.5–6.5 range, but none of isolate could tolerate exposure to pH 1.0.
sakei (GenBank acc #KM267629, KU317778 and LC064899), and L. lactis subsp. cremoris 3-­3 and 1-­5 strains showed resistance to pH
isolate M17 2-­2 showed 100% similarity with L. lactis subsp. lactis 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
Piyatiratitivorakul, 2008; Ringø et al., 2010; Pérez-­Sánchez, Balcázar,
vagococcosis and lactococcosis
Garcia, et al., 2011). The previous studies have shown that antagonis-
Cumulative mortalities after challenging with L. garviae were 10.71, tic effects against L. garvieae of L. lactis subsp. cremoris DSM 20069
24.99 and 53.57% in fish fed diets containing L. sakei 2-­3, L. lac- (Balcázar et al., 2007), L. mesenteroides CLFP 196 and L. plantarum
tis subsp. lactis M17 2-­2 and control group, respectively (Table 3). CLFP (Vendrell et al., 2008) and L. plantarum, L. lactis and L. mesen-
Statistical analysis demonstrated that fish fed with diet containing L. teroides strains (Pérez-­Sánchez, Balcázar, Garcia, et al., 2011) isolated
sakei and L. lactis subsp. lactis M17 2-­2 at 21 days had significantly from the intestines of healthy salmonid fish, Weissella viridescens F1,
(p < .05) lower mortalities than the control group. Relative per cent F4, F9, F10 and F11 strains from Bahia Blanca Estuary (Sica et al.,
survival were thus 80 and 53% in fish fed with diet containing L. sakei 2010), and L. garvieae A7 strain isolated from rainbow trout farm
2-­3 and L. lactis subsp. lactis M17 2-­2, respectively. water (Didinen, Metin, Onuk, Takmaz, & Ersoy, 2014). In addition,
Fish fed diets containing LAB strains showed a cumulative mor- Balcázar et al. (2007) reported inhibitory activity of Lactobacillus cur-
tality ranging from 35.71% (L. lactis subsp. lactis M17 2-­2) to 49.99% vatus (CLFP150) isolated from the intestine of rainbow trout against
(L. garvieae 1-­4) after challenging with V. salmoninarum. Mortality was V. salmoninarum. However, to our best of knowledge, the inhibition of
50% in the control group (Table 3). However, no significant differences V. salmoninarum by Lactococcus lactis subsp. lactis; Lactococcus lactis
(p > .05) were observed among mortalities in all groups. External ex- subsp. cremoris; Lactococcus garvieae; Lactobacillus sakei; Pediococcus
amination and bacteriological analysis of fish that died during the acidilactici isolates and the inhibition of L. garvieae by Lactobacillus
study revealed the presence of L. garvieae and V. salmoninarum in all sakei has not been described before.
cases. 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
4 |  DISCUSSION also reported the pathogenicity of Weissella spp. for rainbow trout
(Figueiredo, Costa, Leal, Carvalho-­Castro, & Leite, 2012; Liu, Li, Ji, &
In the present study, we obtained a pool of bacterial isolates from Yang, 2009), as well as Lactococcus lactis for Atlantic salmon and rain-
rainbow trout and characterized those with inhibitory activity against bow trout (Austin & Austin, 2007).
L. garvieae and V. salmoninarum. Our study demonstrated that 8 The ability to adhere to intestinal epithelial cells is thought to be
(8.16%) of the screened bacteria (n = 98) isolates were antagonistic an important characteristic for potential probiotic strains. Pan, Li, and
to V. salmoninarum and 4 (4.08%) to L. garvieae. These findings sup- Liu (2006) assessed the ability of LAB strains to adhere to intestinal
port the view of other studies claiming that antagonism between en- epithelial cells and concluded that the more hydrophobic strains dis-
dogenous gut microorganisms of fish and bacterial pathogens occurs played stronger adhesive capability. We found that L. lactis subsp. lac-
in nature and the establishment of a normal or protective microbiota tis M17 2-­2, L. lactis subsp. cremoris and L. garvieae strains, L. sakei 2-­3
might constitute a key component of defensive barrier function (Cain and P. acidilactici 3-­5 were hydrophobic. Our results are in agreement
& Swan, 2010; Gómez & Balcázar, 2008; Pérez-­Sánchez, Balcázar, with previous studies, which demonstrated that L. sakei, L. lactis, L.
Garcia, et al., 2011). Indeed, several LAB strains isolated from fish plantarum, L. fermentum and L. mesenteroides isolated from rainbow
and aquatic animals display antagonistic activity against fish patho- trout intestine tended to adhere to intestinal mucosa (Balcázar et al.,
genic agents (Joborn, Olsson, Westerdahl, Conway, & Kjelle-­berg, 2007, 2008; Pérez-­Sánchez, Balcázar, Garcia, et al., 2011). Tolerance
1997; Nikoskelainen et al., 2001; Ringø et al., 2002; Balcázar et al., 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 Austin, B., & Austin, D. A. (2007). Bacterial Fish Pathogens: Diseases of
study, all L. lactis subsp. cremoris strains and L. garvieae 1-­3 were re- Farmed and Wild Fish, 4th ed.. Chichester, UK: Springer-Praxis.
Avcı, H., Aydogan, A., Tanrıkul, T. T., & Birincioğlu, S. S. (2010). Pathological
sistant to 2.5%–10% bile concentrations, while Lactobacillus sakei 2-­3
and microbiological investigations in rainbow trout (Oncorhynchus
was resistant to only 2.5% bile concentration. Similarly, Balcázar et al. mykiss Walbaum, 1792) naturally infected with Lactococcus garvieae.
(2008) noted L. lactis, L. plantarum and L. fermentum can survive at Kafkas Universitesi Veteriner Fakultesi Dergisi, 16, 313–318.
2.5%–10% bile for 1.5 hr. Indeed, the concentration of bile in the in- Balcázar, J. L., Vendrell, D., de Blas, I., Ruiz-Zarzuela, I., Gironés, O., &
Múzquiz, J. L. (2007). In vitro competitive adhesion and production of
testine of salmonid fish has been estimated to be ranging between
antagonistic compounds by lactic acid bacteria against fish pathogens.
0.4 and 1.3% (Balcázar et al., 2008). Thus, Pérez-­Sánchez, Balcázar, Veterinary Microbiology, 122, 373–380.
Garcia, et al. (2011) used 0.2%–1% bile concentrations in their study Balcázar, J. L., Vendrell, D., de Blas, I., Ruiz-Zarzuela, I., Múzquiz, J. L., &
and reported tolerance of L. lactis, L. plantarum ve L. mesenteroides to Gironés, O. (2008). Characterization of probiotic properties of lactic
acid bacteria isolated from intestinal microbiota of fish. Aquaculture,
1.0% bile. The pH of gastric juice is the main factor that determines
278, 188–191.
the survival of bacteria that pass from the stomach to the intestine Brunt, J., & Austin, B. (2005). Use of a probiotic to control lactococcosis
(Balcázar et al., 2008). In this study, Lactococcus lactis subsp. cremoris and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum).
3-­3 and 1-­5, Lactococcus garvieae 1-­3 and P. acidilactici 3-­5 showed Journal of Fish Diseases, 25, 333–342.
Cain, K., & Swan, C. (2010). Barrier function and immunology. In M. Grosell,
resistance to pH 2.5. Similarly, Balcázar et al. (2008) noted L. lactis,
A. P. Farrell, & C. J. Brauner (Eds.), The Multifunctional Gut of Fish (pp.
L. plantarum and L. fermentum can survive at pH 2.5. Pérez-­Sánchez,
111–134). Amsterdam: Academic Press.
Balcázar, Garcia, et al. (2011) also reported that L. lactis, L. plantarum Didinen, B. I., Kubilay, A., Diler, O., Ekici, S., Onuk, E. E., & Findik, A. (2011).
and L. mesenteroides were tolerant at pH 3.0. First isolation of Vagococcus salmoninarum from cultured rainbow
To date, the beneficial effects of probiotic administration against trout (Oncorhynchus mykiss, Walbaum) broodstocks in Turkey. Bulletin-­
European Association of Fish Pathologists, 31, 235–243.
L. garvieae infection in rainbow trout have been demonstrated by in-
Didinen, B. I., Metin, S., Onuk, E. E., Takmaz, H., & Ersoy, A. T. (2014).
corporation of Aeromonas sobria GC2 (Brunt & Austin, 2005), L. mes- Isolation and characterization of potential probiotic bacteria from rain-
enteroides CLFP 196, L. plantarum CLFP 238 (Vendrell et al., 2008) and bow trout Oncorhynchus mykiss,(Walbaum) rearing units against bac-
L. plantarum (Pérez-­Sánchez, Balcázar, Merrifield, et al., 2011). To our terial pathogens. The Israeli Journal of Aquaculture-­Bamidgeh, 66, 1–6.
Diler, O., Altun, S., Adıloglu, A. K., Kubilay, A., & Isıklı, B. I. (2002). First
knowledge, however, this is the first report describing the probiotic
occurence of streptococcosis affecting farmed rainbow trout (O. my-
effect of endogenous L. lactis subsp. lactis M17/2-­2 and L. sakei 2-­3 kiss) in Turkey. Bulletin-­European Association of Fish Pathologists, 22,
against L. garvieae infection in rainbow trout. 21–26.
As a conclusion, LAB isolates from rainbow trout intestines could Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using
the bootstrap. Evolution, 39, 783–791.
serve as a potential probiotics with ability to inhibit pathogen growth
Figueiredo, H. C. P., Costa, F. A. A., Leal, C. A. G., Carvalho-Castro, G. A., &
under in vitro conditions, acid and bile tolerance as well as adhesion Leite, R. C. (2012). Weissella sp. outbreaks in commercial rainbow trout
to intestinal mucus. Moreover, L. lactis subsp. lactis M17/2-­2 and L. (Oncorhynchus mykiss) farms in Brazil. Veterinary Microbiology, 156(3),
sakei 2-­3 strains inhibited growth of L. garvieae and V. salmoninarum. 359–366.
Ghittino, C., Accornero, P., Prearo, M., Rogato, F., Zlotkin, A., & Eldar, A.
Although these strains decreased mortality caused from lactococcosis,
(1999). Coldwater streptococcosis in salmonids, with particular refer-
no significant effect was observed for vagococcosis. Results suggest ence to Vagococcus salmoninarum infection. Proceedings of Workshop
that L. lactis subsp. lactis M17/2-­2 and L. sakei 2-­3 strains can provide in Fish Streptococcoses, IZS – State Veterinary Institute, Turin, Italy.
a viable alternative for managing lactococcosis in cultured fish. Gómez, G. D., & Balcázar, J. L. (2008). A review on the interactions be-
tween gut microbiota and innate immunity of fish. FEMS Immunology
and Medical Microbiology, 52, 145–154.
Hjelm, M., Bergh, O., Riaza, A., Nielsen, J., Melcheiorsen, J., Jensen, S., …
ACKNOWLE DG E MEN T
Gram, L. (2004). Selection and identification of autochthonous poten-
This work has been partly supported by Scientific and Technological tial probiotic bacteria from turbot larvae (Scophthalmus maximus) rear-
ing units. Systematic and Applied Microbiology, 27, 360–371.
Research Council of Turkey (TUBITAK) under Project No: 113 O 617
Irianto, A., & Austin, B. (2002). Use of probiotics to control furunculosis in
and partly supported by Suleyman Demirel University BAP (Scientific rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases,
Research Projects Coordination Unit) under Project No: SDU-­BAP 25, 333–342.
-­3740-­YL2-­13. Joborn, A., Olsson, J. C., Westerdahl, A., Conway, P. L., & Kjelle-berg, S.
(1997). Colonization in the fish intestinal tract and production of
inhibitory substances in intestinal mucus and faecal extracts by
Carnobacterium sp. strain K1. Journal of Fish Diseases, 20, 383–392.
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