isolamento de probióticos mourio2016

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Aquaculture Reports 3 (2016) 166–171
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Aquaculture Reports
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Isolatio So
Pseudo ma
(Silurif l ap
José Luiz elip
Adolfo B orr
Walter Q , Ma
a AQUOS—Labo ade F
Gonzaga 1346,
b Laboratório d
c Instituto Federal Catarinense (IFC), Campus Araquari., BR 280, Km 27, 89245-000, Araquari, SC, Brazil
d Unidade de Reproduc¸ ão Mar e Terra, Rod. BR 364, Linha 36, Km 01, 76970-000 Pimenta Bueno, RO, Brazil
e Centro de Desenvolvimento em Aquicultura e Pesca, Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina (EPAGRI), Rua Admar Gonzaga
1188, 88010-970 Florianópolis, SC, Brazil
a r t i c l
Article history:
Received 20 Ju
Received in re
Accepted 3 Ma
Keywords:
Fish nutrition
Sorubim
Lactic acid bac
Weissela cibari
1. Introdu
Fish of th
Paraguay Ri
didae famil
2003) and 
et al., 2003)
the United 
American c
about 670 t
∗ Correspon
E-mail add
http://dx.doi.o
2352-5134/© 
0/).
 e i n f o
ly 2015
vised form 23 February 2016
rch 2016
teria
a
a b s t r a c t
This study investigated bacterial strains with probiotic potential isolated from the middle portion
of healthy hybrid surubim catfish foregut (Pseudoplatystoma reticulatum female × P. corruscans male).
Twenty surubims weighing 1.5 ± 0.3 kg were used for bacterial isolation. In total, 41 strains of bacteria
were selected in vitro. Ten strains had inhibition zones >10 mm against Aeromonas hydrophila. Five of those
strains presented inhibition zones > 9 mm against other pathogenic bacteria and reached concentrations
greater than 105 CFU mL−1 in tubes containing de Man, Rogosa and Sharpe (MRS) medium. In particular,
Weissella cibaria (P36) reached 106 CFU mL−1 in MRS and was able to reduce the pH of the medium to
3.85. In the in vivo intestinal colonization studies, 72 healthy hybrid surubims were fed with a commercial
diet supplemented with probiotic W. cibaria for 15 days. Changes in gut community composition were
then analyzed, and probiotic profile of W. cibaria was determined molecularly by amplification of rRNA
16S gene was performed using PCR. Compared to control fish, W. cibaria-supplemented fish showed an
increase in RBC. These results show the efficacy of our haematological approach to probiotic screening
in hybrid sorubim.
© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
ction
e genus Pseudoplatystoma found in the Uruguay River,
ver and São Francisco River basins belong to the Pimelo-
y. They can reach 152 cm in length (Agostinho et al.,
are of great economic importance in Brazil (Roubach
. According to the Food and Agriculture Organization of
Nations (FAO, 2009), aquaculture production of South
atfish species in continental waters in 2007 reached
ons, producing revenue close to $1,467,000.00 USD.
ding author.
ress: gabriel faj@hotmail.com (G.F.A. Jesus).
However, as reported by Moraes and Martins (2004), high pro-
duction results in high stock density and equally high incidence of
disease. In particular, diseases of bacterial origin cause the great-
est economic losses, often manifesting as subclinical changes in
infected fish (Martins et al., 2004). For example, the bacterial genus
Aeromonas caused primary and secondary septicemia in immuno-
compromised hybrids of Pseudoplatystoma species, as determined
in different aquaculture outbreaks in Mato Grosso, Brazil (data not
shown).
Antibiotics have typically been used to prevent and control dis-
ease in intensive fish farming. However, studies have reported an
increase in resistance to pathogens and growth in aquaculture
systems (Verschuere et al., 2000). Therefore, the use of probi-
otics is now considered a viable prophylactic alternative. Probiotics
are defined as live organisms that benefit the health of the host
rg/10.1016/j.aqrep.2016.03.001
2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.
n of probiotic bacteria from the hybrid
platystoma reticulatum × Pseudoplatysto
ormes: Pimelodidae): A haematologica
 Pedreira Mourin˜oa,b, Gabriella do Vale Pereiraa, F
ezerra Jatobáa,c, Thiago Tetsuo Ushizimad, Bruno C
uadros Seiffertb, Gabriel Fernandes Alves Jesusa,∗
ratório de Sanidade de Organismos Aquáticos, Departamento de Aquicultura, Universid
 88034-001 Florianópolis, SC, Brazil
e Camarões Marinhos, UFSC, Beco dos Coroas 503, 88061-600 Florianópolis, SC, Brazil
uth American catfish
 corruscans
proach
e do Nascimento Vieirab,
ea da Silvaa,e,
urício Laterc¸ a Martinsa
ederal de Santa Catarina (UFSC), Rod. Admar
J.L.P. Mourin˜o et al. / Aquaculture Reports 3 (2016) 166–171 167
Table 1
Antagonistic activitya of lactic acid bacteria isolated from the intestine of healthy hybrid surubim (Pseudoplatystoma reticulatum female x P. corruscans male) against Aeromonas
hydrophila subsp. hydrophila (CPQBA 228–08 DRM).
Strains Morphology Gram Inhibitionzone (mm) Strains Morphology Gram Inhibitionzone (mm)
P1 
P2 
P3 
P4 
P5 
P6 
P7 
P8 
P9 
P10 
P11 
P12 
P13 
P14 
P15 
P16 
P17 
P18 coccus + 9.33
P19 coccus + 0.00
P20 coccus + 9.00
P21 
a Determine obiotic bacterial strain using the WDA method.
Table 2
Antagonist act doplatystoma reticulatum female x P. corruscans male) against Aeromonas
hydrophila sub us (VA), Vibrio anguillarum (VAN), Enterococcus durans (ED), Pseudomonas
aeruginosa (PA io cholera (VC).
Strain 
P2 
P4 
P6 
P13 
P14 
P28 
P29 
P33 
P35 
P36 
a Determine
when admi
benefits inc
duction of b
(De et al., 2
haemato-im
Mourin˜o et
lactic acid b
(Oreochrom
in the immu
cus durans.
This stud
the intestin
Linnaeus 17
to determin
nal epitheli
haematolog
2. Materia
2.1 Selec
For lacti
weighing 1.
dos, MS, Br
with chemi
0.01% benzo
bacillus + 0.00 P22 
bacillus coccus + 10.00 P23 
coccus + 0.00 P24 
bacillus + 10.00 P25 
bacillus + 9.33 P26 
bacillus + 10.50 P27 
coccus + 0.00 P28 
coccus + 0.00 P29 
coccus + 5.33 P30 
coccus + 0.00 P31 
Bacillus coccus + 8.33 P32 
coccus + 0.00 P33 
bacillus + 10.67 P34 
coccus + 12.33 P35 
coccus + 0.00 P36 
coccus + 9.33 P37 
coccus + 8.33 P38 
coccus + 0.00 P39 
coccus + 0.00 P40 
coccus + 3.00 P41 
coccus + 8.67
d by the diameter of the inhibition zone formed around the discs containing the pr
ivitya of lactic acid bacteria isolated from the intestine of hybrid surubim (Pseu
sp. hydrophila (AH), Aeromonas sobria (AS), Vibrio harveyi (VH), Vibrio alginolytic
), Escherichia coli (EC), Micrococcus luteus (ML), Staphylococcus aureus (SA), and Vibr
Inhibition zone (mm)
Species AH AS PA ED VC 
N 7.83 6.00 6.00 6.00 11.33 
Lactobacillus confusus 9.00 8.00 8.17 6.00 10.33 
Lactobacillus brevis 11.33 8.83 8.17 6.00 10.17 
Lactobacillus brevis 9.00 8.00 10.00 6.00 14.50 
Lactococcus lactis lactis 8.33 6.00 8.33 6.00 9.83 
N 8.33 6.00 8.00 6.00 9.33 
Lactococcus lactis lactis 9.33 6.00 15.00 6.00 11.33 
Lactobacillus brevis 10.67 6.00 9.17 8.33 11.33 
Lactobacillus confusus 9.00 6.00 6.00 6.00 13.83 
Weissella cibaria 9.33 6.00 8.67 6.00 14.50 
d by the diameter of the inhibition zone formed around the discs containing the probiot
nistered in adequate concentrations (Reid, 2008). Such
lude competitive exclusion of pathogenic bacteria, pro-
acteriocins and organic acids, and immunomodulation
014). Indeed, the use of probiotics has improved the
munological parametersin fish (Barbosa et al., 2011;
 al., 2012; Ridha and Azad 2012). For example, using
acteria isolated from the intestinal tract of Nile tilapia
is niloticus). Jatobá et al. (2008) described improvement
ne system after experimental infection with Enterococ-
y aimed to investigate strains of bacteria isolated from
al tract of hybrid catfish (Pseudoplatystoma reticulatum
66 female × P. corruscans Spix and Agassiz 1829 male)
e probiotic potential, ability to adhere to the intesti-
um, as well as modify host microbiota and improve
ical parameters.
ls and methods
tion of probiotic strains
c acid bacterial isolation, 20 healthy hybrid surubims
5 ± 0.3 kg were obtained from Mar & Terra Ltda (Doura-
azil). They were reared in tanks not previously treated
cals or antibiotics. The animals were anaesthetized with
caine. Their intestines were excised and then washed
Table 3
Incorporation
of hydrogen io
Strains 
Lactobacillus
Lactobacillus
Lactococcus 
Lactobacillus
Weissella cib
with sterile
All animal p
CEAU/UFSC
sue was the
ratio with t
Man, Rogos
were incub
MRS plates
characteris
The foll
in vitro fun
hydrophila 
symptomat
cus (CPQBA
Marguerita
coccus + 8.50
coccus + 0.00
coccus + 0.00
coccus + 5.33
coccus + 8.50
bacillus coccus + 5.33
bacillus coccus + 12.00
coccus + 10.33
bacillus coccus + 0.00
bacillus coccus + 0.00
coccus + 0.00
bacillus + 14.00
coccus + 0.00
bacillus + 10.33
bacillus + 11.00
coccus + 8.50
coccus + 0.00
EC ML SA VA VAN VH
6.00 9.33 6.00 6.00 6.50 5.50
10.00 8.50 9.00 7.00 6.60 7.40
10.33 9.00 10.00 6.90 8.33 8.20
10.33 16.50 8.17 8.00 7.50 6.50
8.33 14.50 8.00 6.30 6.00 6.00
6.00 6.00 6.00 7.33 5.33 6.00
8.33 13.00 8.33 7.50 6.33 6.00
10.33 14.00 7.67 7.33 11.30 8.33
9.17 13.67 6.00 7.40 8.00 9.20
11.83 11.33 8.67 8.33 11.25 10.00
ic bacterial strains using the WDA method.
 of different lactic acid bacteria in the commercial diet and evaluation
n dissociation (pH) in MRS media at 35 ◦C for 48 h.
MRS media (CFU/mL) pH Diet (CFU/g)
 brevis (P6) 1.20 × 105 4.50 1.20 × 103
 brevis (P13) 5.10 × 106 4.40 2.10 × 104
lactis lactis (P29) 2.20 × 105 4.13 1.20 × 104
 brevis (P33) 2.12 × 106 4.20 1.20 × 104
aria (P36) 1.00 × 107 3.85 1.00 × 106
 0.65% saline solution to remove non adherent bacteria.
rocedures were approved by the Ethics Committee of
 (Protocol # 23080.021883/2009-91). The excised tis-
n homogenized in sterile 0.65% saline solution at a 1:1
he sample. The supernatant was removed and spread on
a and Sharpe (MRS) Agar (HIMEDIA®) plates. The plates
ated at 35 ◦C for 48 h. Colonies were again spread on
 for an additional 48 h to ensure purity and confirm the
tic morphology of Lactobacillus by Gram staining.
owing pathogenic bacterial strains were used for
ctional antagonist assay: Aeromonas hydrophila subsp.
(CPQBA 228–08 DRM), Aeromonas sobria (isolated from
ic fish), Vibrio harveyi (ATCC 14126), Vibrio alginolyti-
 378–12 DRM), Vibrio anguillarum (donated by Dr.
 Barraco − UFSC), Enterococcus durans (ATCC 19492),
168 J.L.P. Mourin˜o et al. / Aquaculture Reports 3 (2016) 166–171
Table 4
Different genera of bacteria expressed in bacterial concentration on the log scale of CFU per gram of hybrid surubim (Pseudoplatystoma reticulatum female × P. corruscans
male) intestine supplemented and unsupplemented with Weissella cibaria (P36) in the diet. The values of bacterial concentration are expressed in CFUs per gram of intestine
(log (x + 1) CFU g−1).
Treatments ia 
Unsuppleme
Supplement
Different lette ented
Pseudomona
Micrococcus
Vibrio algin
For bact
vated, cultu
30 ◦C for 24
species-spe
of economi
first evalua
according t
(2006). Isol
as measure
After incub
hydrophila 
MRS plates 
agar and inc
sured by th
placed in tr
Ten stra
hydrophila 
Kit (API, bio
bacteria we
The results
bioMérieux
10 strains b
tial were re
To interp
in different 
(level 3); 
tal waters 
in marine 
pathogens 
NaCl. All ba
Gram staini
Five sele
ity assays a
fish with 45
acidifying t
tion of diss
determine 
MRS mediu
tration of d
strain, assu
peroxide ca
Next, W
ing of the 
25.00 g kg−1
10.00 g kg−1
(max) 100.0
ity (max) 12
diet as a sp
diet was in
samples of s
1.5% saline s
of 10−5 and
for 48 h to e
ologic
uscan
 the d
lls (W
ls (BA
mpho
eters 
106 �
103 �
x103 �
)
103 �
103 �
103 �
103 �
103 �
x103 �
g dL−
t lette
upplem
as sp
m, bu
testin
enty
 wei
ross
ct col
4 h s
First, the intestinal tract was extracted with tweezers and
l, washed twice with sterilized water, and homogenized in
ar with sterile saline solution serially diluted (1:10). Next,
lution was spread on TSA agar for total heterotrophic bac-
ount, MRS agar to determine lactic acid bacteria, Cetrimide
r Pseudomonas sp. selection, and TCBS agar for Vibrionaceae
on. These were all incubated at 30 ◦C. Total CFU counts were
ined after 24 h of incubation in TSA, Cetrimide, and TCBS
 and 48 h in MRS. The colonies grown on MRS agar were
stained and selected for subsequent reisolation and bio-
al characterization (API test; bioMérieux) to confirm the
ation and composition of host bacteria.
perimental design
elve circular 100 L tanks with aeration and constant heating
 were used. The biological filtration system (gravel biofil-
as independently maintained to ensure water quality (pH
). Each day, sedimentary deposits and 70% of the water were
ed from the tanks. Water quality was measured as follows:
Totalheterotrophic Lactic acid bacter
nted fish 6.58 ± 1.13a 0.60 ± 1.04a
ed fish 5.71 ± 0.14a 3.72 ± 0.98b
rs indicate statistical significant difference between supplemented and unsupplem
s aeruginosa (ATCC 27853), Escherichia coli (D363),
 luteus (A270), Staphylococcus aureus (ATCC 29213), and
olyticus (isolated by Martins et al., 2010).
erial inhibition, the strains noted above were reacti-
red, and maintained in Tryptic Soy Agar (TSA, Oxoid®) at
 h. For in vitro selection of antagonists, Pseudoplatystoma
cific pathogens were chosen, as well as other pathogens
c importance to aquaculture. The isolated strains were
ted for inhibition of A. hydrophila (CPQBA 228–08 DRM)
o the wet disc assay (WDA) described by Ramiréz et al.
ates were inoculated in MRS agar plates at 108 CFU/mL,
d by spectrophotometry, and incubated at 35 ◦C for 24 h.
ation, 1 cm discs were removed from the plates, and A.
was inoculated on TSA agar plates. The discs from the
were placed on top of the recently inoculated pathogen
ubated at 30 ◦C for 24 h. Antagonistic activity was mea-
e diameter (mm) of the inhibition zone around the discs
iplicate on each plate.
ins with the greatest antagonistic capacity against A.
were phenotypically characterized using the API 50CH
Mérieux, Hazelwood, MO, USA). Lactic acid-consuming
re identified by the fermentation of 49 carbohydrates.
 were interpreted according to APIWEB software (API,
, Hazelwood, MO, USA). Following evaluation of these
y WDA, only five with the greatest antimicrobial poten-
tained.
ret the inhibitory results, the pathogens were assigned
levels of importance: specific pathogens for aquaculture
nonspecific pathogens for aquaculture in continen-
(level 2), and nonspecific pathogens for aquaculture
waters (level 1). To assess the inhibition of marine
(Vibrio spp.) by WDA, TSA agar was enriched with 2%
cterial cultures were routinely checked for purity by
ng and depletion on plates.
cted pathogenic strains were then submitted to viabil-
fter inoculation in a commercial diet for carnivorous
% crude protein. The ability to produce organic acids by
he culture medium was determined by the concentra-
ociated hydrogen ions (pH). The strains were tested to
colony forming units (CFU) per mL in tubes containing
m (pH 6) at 35 ◦C for 48 h. After incubation, the concen-
issociated hydrogen ions (pH) wasmeasured for each
ming that the production of organic acids or hydrogen
used any changes in the values (Vazquez et al., 2005).
. cibaria was added in a commercial diet consist-
following recommended ingredients: calcium (max)
, ether extract (min) 100.00 g kg−1, phosphorus (min)
, fibrous material (max) 15.00 g kg−1, mineral material
0 g kg−1, crude protein (min) 400.00 g kg−1, and humid-
0.00 g kg−1. A bacterial solution was then applied to the
ray at a ratio of 200 mL kg−1. After incorporation, the
Table 5
Haemat
x P. corr
(P36) in
blood ce
basophi
(NEU), ly
Param
RBC (x
TTC (x
WBC (
HTC (%
BAS (x
EOS (x
PAS (x
NEU (x
LIM (x
MON (
GLC (m
Differen
and uns
diet w
mediu
2.1. In
Sev
65 g in
Mato G
nal tra
After 2
dures. 
scalpe
a mort
the so
terial c
agar fo
selecti
determ
media
Gram-
chemic
coloniz
2.2. Ex
Tw
(26 ◦C)
ters) w
6.8–7.2
remov
cubated with air circulation at 45 ◦C for 18 h. Three 1 g
upplemented dry diet were macerated in 1 mL of sterile
olution and serially diluted (1/10) nine times. Dilutions
 10−9 were spread on MRS agar and incubated at 35 ◦C
stimate a desirable concentration 106 CFU g−1. Control
pH 6.8 ± 0.2
8.5 mg/L. Si
days. After 
twice a day
were rando
supplemen
Pseudomonas spp. Enterococcus spp.
5.63 ± 1.95a 3.38 ± 0.45a
4.76 ± 0.72a 2.27 ± 0.07b
 fish by t-test (p < 0.05).
al parameters of hybrid surubim (Pseudoplatystoma reticulatum female
s male) supplemented and unsupplemented with Weissella cibaria
iet at a concentration of 107 CFU.g−1. Red blood cells (RBC), white
BC), total thrombocytes count (TTC), haematocrit percentage (HTC),
S), eosinophils (EOS), granular leukocyte PAS+ (PAS), neutrophils
cytes (LIM), monocytes (MON), and glucose levels (GLC).
Supplemented fish Unsupplemented fish
L−1) 1.85 ± 1.6a 1.57 ± 1.5 b
L−1) 50.84 ± 6.3 a 36.72 ± 14.0 a
L−1) 52.34 ± 6.9 a 44.41 ± 15.2 a
24 ± 4.9 a 23.2 ± 6.7 a
L−1) 0 0
L−1) 0.3 ± 0.1 a 0.75 ± 0.75 a
L−1) 0.99 ± 0.8 a 1.29 ± 0.4 a
L−1) 2.66 ± 1.8 a 2.22 ± 2.3 a
L−1) 38.34 ± 14.0 a 34.83 ± 17.1 a
L−1) 3.48 ± 1.9 a 2.15 ± 1.6 a
1) 47.27 ± 12.7 a 30.72 ± 9.3 a
rs indicate statistical significant difference between supplemented
ented fish (p < 0.05).
rayed with the same concentration of sterilized MRS
t without bacteria.
al tract colonization
-two healthy hybrids approximately 21 cm in length and
ght were obtained from Mar & Terra Ltda (Dourados,
o do Sul, Brazil). These fish were used to evaluate intesti-
onization by the W. cibaria selected in the previous step.
tarvation, the next steps included the following proce-
; total ammonia 1.2 ± 0.4 mg/L; and dissolved oxygen
x fish were placed in each tank and acclimated for 5
this period, they were fed with the experimental diets
 for 15 days or unsupplemented diets (control). Fish
mly distributed in tanks divided into Weissela cibaria-
ted fish and control fish in 6 replicates.
J.L.P. Mourin˜o et al. / Aquaculture Reports 3 (2016) 166–171 169
2.3. Sampling
At the end of the experiment, the fish from each experimental
unit were sampled. Following anesthesia with eugenol (75 mg/L),
blood was 
syringes, on
one withou
ogy analysi
2 h at 25 ◦C
and stored 
2.4. Haema
Blood sm
ferential co
leukocytes 
hematocrit 
in ice to co
Neubauer c
was used to
ter (Spectru
Kit (Biotecn
2.5. Statisti
Data we
Bartlett test
the data we
counts and 
2.6. Molecu
After the
at the State
DNA extrac
performed 
p1401r hom
of bacteria.
Amplific
an automat
primers us
sequences 
ent primer
sequences o
DNA seque
(Thompson
using the M
tance matri
The phylog
tances by th
bootstrap v
software.
3. Results
3.1. Selectio
A total o
ical charact
demonstrat
(Table 1). Th
Lactobacillu
Weissella cib
Kit (bioMér
Strains t
other patho
6
98
80
Weissella koreensis S-5623 (AY03 5891)
Weissella kandleri DSM 20593 (M23038)
hylog
A 001
s gen
L. bre
d a m
er, o
educ
testin
 fed
ia (3.
1 CF
 viab
 still
 in K
U g−1
aema
 blo
d w
eters
olecu
 stra
 the microorganism collection at the Multidisciplinary Cen-
Chemistry, Biology and Agriculture Research (CPQBA) of the
niversity of Campinas (UNICAMP).
cussion
s study investigated bacterial strains with probiotic potential
d from the mid-foregut of healthy hybrid surubim cat-
seudoplatystoma reticulatum female x P. corruscans male).
ne strains of bacteria were isolated in this study. Seven
strated inhibition zones >8 mm against pathogenic bacte-
her studies have isolated bacterial strains with probiotic
ial. For example, Huys et al. (2001) and Fjellheim et al.
 isolated 120 and 500 bacterial strains from Atlantic cod
 morhua) and turbot (Scophthalmus maximus), respectively.
g et al. (2010) evaluated the in vitro antagonism of pro-
bacteria against common pathogens in the Atlantic cod.
iscovered dissimilarities in inhibition capacity among these
. Pan et al. (2008) also evaluated the in vitro antagonism of
collected from the caudal vessel using two 21 G 3 mL
e containing a drop of 10% EDTA as anticoagulant and
t. The blood with anticoagulant was used for haematol-
s. The blood without anticoagulant was incubated for
, centrifuged at 1400g for 10 min to obtain the serum,
at −20 ◦C for glucose analysis.
tology
ears were stained by Giemsa/May Grunwald for dif-
unt of leukocytes and total count of thrombocytes and
(WBC) (Ishikawa et al., 2008). An aliquot was used for
determination, and the remaining blood was stored
unt the total number of erythrocytes (RBC) using a
hamber (Ranzani-Paiva et al., 2013). An aliquot of serum
 determine glucose concentration by spectrophotome-
m SP-2100) at 505 nm according to Biotecnica Glucose
ica, Minas Gerais, Brazil).
cal analysis
re evaluated according to variance homogeneity by the
. In cases where variance heterogeneity was observed,
re transformed to log (x + 1) for both the microbiological
blood counts and analyzed by t-test (p < 0.05).
lar identification
 experiment, the bacteria were molecularly identified
 University of Campinas (UNICAMP). Using genomic
ted from the samples, rRNA 16S gene amplification was
with PCR. Primers used for PCR reaction were p27f and
ologous ends of the conserved 16S ribosomal RNA gene
ation products were purified and sequenced using
ed sequencer (MegaBACE 1000, GE Healthcare). The
ed were p10f, 765f, 782r and p1100r. The partial
of the 16S ribosomal RNA gene obtained from differ-
s were assembled into a contig and compared with
f organisms found in the Genbank and RDP databases.
nces were aligned using the CLUSTAL X program
 et al., 1997), and phylogenetic analyses were conducted
EGA 4.0 program (Tamura et al., 2007). Evolutionary dis-
x was calculated based on the model of Kimura (1980).
enetic tree was constructed from the evolutionary dis-
e neighbor-joining method (Saitou and Nei, 1987) with
alues calculated from 1000 recounts using MEGA 4.0
n of probiotic strains
f 41 bacterial strains were isolated, all with morpholog-
eristics of Gram-positive lactic acid bacteria. Only 10
ed inhibition zones > 10 mm, as determined by WDA
ey were identified as Lactobacillus confusus (2 strains),
s brevis (3 strains), Lactococcus lactis lactis (2 strains),
aria (1 strain), and two unidentified strains by API 50CH
ieux, Marcy l’Etoile, France).
hat presented average inhibition zones >9 mm against
gens were L. brevis (P6), L. brevis (P13), L. lactis lactis
46
5
Fig. 1. P
of CPQB
database
(P29), 
reache
Howev
ble of r
3.2. In
Fish
bacter
fish (10
remain
cibaria
grown
102 CF
3.3. H
Red
mente
param
3.4. M
The
kept in
ter for 
State U
4. Dis
Thi
isolate
fish (P
Forty-o
demon
ria. Ot
potent
(2010)
(Gadus
Caipan
biotic 
They d
strains
99
71
98
99
Weilss ell a soli DSM 14420 (AY02826 0)
Weissell a confusa DSM 201 96 (M23036)
Weiss ell a cibaria CHJ3 (AF312874) 
COQBA 001-10 DRM 02
Weissella hellencia NCFB 2873 (X959810) 
Weiss ell a thail andensis FS 61-1 (ABO23838)
Weiss ell a parameseneroides DMS-20288 (M23033)
Weiss ell a videsce ns DMS-20410 (M23040)
enetic relationship among the sequences of 16S ribosomal RNA gene
–10 DRM 02 and other microorganisms presented in the related
bank and RDP.
vis (P33), and W. cibaria (P36) (Table 2). These strains
inimum concentration of 103 CFU.mL−1 in the diet.
nly W. cibaria (P36) reached 106 CFU.mL−1and was capa-
ing the pH of MRS to 3.85 (Table 3).
al tract colonization
 W. cibaria showed a higher value of total lactic acid
72 × 104 CFU g−1) in MRS compared to unsupplemented
U g−1). These results demonstrated that W. cibaria could
le in the intestine of supplemented fish (Table 4). W.
 reduced the bacterial load of Streptococcus selectively
F agar. Supplemented fish showed a concentration of
bacteria; control fish presented 104 CFU g−1 bacteria.
tology
od cell count significantly increased in fish supple-
ith W. cibaria (p < 0.05). The other haemotological
 did not statistically differ (Table 5).
lar identification
in of W. cibaria was molecularly confirmed (Fig. 1) and
170 J.L.P. Mourin˜o et al. / Aquaculture Reports 3 (2016) 166–171
Clostridium butyricum CB2c, as well as its ability to adhere to intesti-
nal cells and inhibit the growth of A. hydrophila and V. anguillarum.
Other investigators have focused on the antagonism of probiotics
isolated from marine organisms. For instance, Jatobá et al. (2008)
isolated L. 
Lactobacillu
present stu
to 3.85. Th
pH in vivo a
confirms th
peroxide to
Apart fro
terion for th
in the intes
biotic bacte
the intestin
2001). In pa
tive tract to
study, W. cib
and lost on
Vendrell et
Similar resu
colonizing t
the diet (10
Jatobá et al.
domonas co
concentrati
There is
high concen
health (Gom
supplemen
compared t
L. plantarum
with a symb
Increased v
Pedro et al.
culatory su
Very im
profile. Acc
Lactobacillu
organisms a
(Nikoskelai
ferred by L. 
supplemen
method in 
absence of f
acid bacter
have been c
pathogenic
investigate 
nal microbi
the diet (Yo
5. Conclus
This stud
from the in
promising b
Acknowled
The auth
nological D
support and
istry of Fish
Mar & Terra Ltda for fish donation; Drs. E. Zaniboni Filho, R.Y. Fuji-
moto, A.R.M. Magalhães, W.Q. Seiffert and R.P. Schocken-Iturrino
for critical review of the manuscript.
nces
o, A.A
er par
.), Mig
us. Vit
, M., Ja
ivatio
iotic i
, C.M.A
erial i
vestig
256.
o, N., G
. Dail
meter
 Meen
iotics
hysio
9. Foo
ons fo
, A.J.,
ndida
us mo
G.D., B
obiot
154.
, Dher
ficial 
acultu
a, N.M
hods i
., Viei
ic-acid
iotic. 
., Ols
nizati
testin
20 (5)
 M., 19
titutio
. 16, 1
, M.L., 
atolo
eichth
esse d
, M.L., 
bá, A.,
olytic
eichth
 F.R., M
cipais
., Frac
gua do
, J.L.P
. Effe
ato-i
acult. 
lainen
bow tr
nosus
lainen
une e
iotic b
452.
Wu, T.
rence
ricum
nchez
, I., 20
orhyn
ieae in
, C., Ci
oorga
as de c
 70–7
-Paiva
ise he
 2008.
11), 9
plantarum from tilapia. Vazquez et al. (2005) isolated
s sp. from rainbow trout (Oncorhynchus mykiss). In the
dy, Weissella cibaria was able to reduce the pH of MRS
e mode of action of W. cibaria in reducing intestinal
nd in vitro remains unknown. Nonetheless, this result
e possibility of producing organic acids and hydrogen
 inhibit Gram-positive bacteria.
m their inhibitory capabilities, the most important cri-
e selection and use of probiotic bacteria is colonization
tinal tract (Verschuere et al., 2000). Specifically, pro-
ria must be able to tolerate bile acids and adhere to
al epithelium (Joborn et al., 1997; Nikoskelainen et al.,
rticular, isolated microbiota should colonize the diges-
 a greater degree than commercial strains. In the present
aria reached values of 3.7 × 104 CFU g−1 in the intestine
ly two orders of magnitude when analyzed in the diet.
 al. (2008) counted 106 CFU g−1 of probiotic in the diet.
lts were found by Jatobá et al. (2008) for L. plantarum
he digestive tract of Nile tilapia (106 CFU mL−1) and in
8 CFU mL−1). However, in contrast to that reported by
 (2008), W. cibaria did not provoke a reduction in Pseu-
ncentration, but rather a decrease of Enterococcus spp.
on.
 ample evidence that probiotic bacteria can reach
trations in the intestinal tract, thus benefiting animal
ez and Balcazar, 2008). In the present study, probiotic-
ted fish showed a significant increase in RBC count
o control fish. This was also reported in Nile tilapia fed
 (Jatobá et al., 2008) and in Pseudoplatystoma sp. fed
iotic diet (inulin and W. cibaria) (Mourin˜o et al., 2012).
alues of RBC improve oxygen supply to fish tissues (De
, 2005), which could account for the more efficient cir-
pply in supplemented fish.
portant consideration in probiotic selection is safety
ording to Nikoskelainen et al. (2001), members of the
s genus have been used in food products as probiotic
nd are usually considered safe. Indeed, previous studies
nen et al., 2003) reported improved immunity con-
rhamnosus JCM 1136. This result indicates that probiotic
tation can be an effective biotherapy or prophylactic
aquaculture. Perez-Sanchez et al. (2011) observed the
urunculosis symptoms in rainbow trout fed with lactic
ia (L. lactis, L. plantarum, L. fermentum). These bacteria
haracterized as probiotics able to inhibit the growth of
 bacteria. However, more studies should be conducted to
the use of food additives relative to altering host intesti-
ota and increasing lactic acid bacteria concentration in
usefian and Amiri, 2009).
ions
y established the efficacy of isolating probiotic bacteria
testine of the hybrid surubim and shows W. cibaria as a
acterium for use in surubim aquaculture.
gements
ors thank the National Council for Scientific and Tech-
evelopment (CNPq/MAPA 577657/2008-9) for financial
 the grant to M.L. Martins (CNPq 302493/2010-7); Min-
ing and Aquaculture of the Federal Republic of Brazil
Refere
Agostinh
upp
(Eds
Stat
Barbosa
Cult
prob
Caipang
bact
an in
249–
De Pedr
1758
para
De, B.C.,
Prob
ecop
FAO, 200
Opti
Fjellheim
of ca
(Gad
Gomez, 
micr
145–
Huys, L.
bene
Aqu
Ishikaw
met
Jatobá, A
Lact
prob
Joborn, A
Colo
in in
Dis. 
Kimura,
subs
Evol
Martins
Hem
(Ost
estr
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Jato
algin
(Ost
Moraes,
prin
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de á
Mourin˜o
2012
haem
Aqu
Nikoske
rain
rham
Nikoske
Imm
prob
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buty
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	Isolation of probiotic bacteria from the hybrid South American catfish Pseudoplatystoma reticulatum×Pseudoplatystoma corru...
	1 Introduction
	2 Materials and methods
	2.1 Intestinal tract colonization
	2.2 Experimental design
	2.3 Sampling
	2.4 Haematology
	2.5 Statistical analysis
	2.6 Molecular identification
	3 Results
	3.1 Selection of probiotic strains
	3.2 Intestinal tract colonization
	3.3 Haematology
	3.4 Molecular identification
	4 Discussion
	5 Conclusions
	Acknowledgements
	References