Aquaculture Reports 3 (2016) 166171

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Aquaculture Reports
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Isolation of probiotic bacteria from the hybrid South American catsh

Pseudoplatystoma reticulatum Pseudoplatystoma corruscans
(Siluriformes: Pimelodidae): A haematological approach
a,b , Gabriella do Vale Pereira a , Felipe do Nascimento Vieira b ,
Jos Luiz Pedreira Mourino
a,c
Adolfo Bezerra Jatob , Thiago Tetsuo Ushizima d , Bruno Correa da Silva a,e ,
Walter Quadros Seiffert b , Gabriel Fernandes Alves Jesus a, , Maurcio Laterca Martins a
a
AQUOSLaboratrio de Sanidade de Organismos Aquticos, Departamento de Aquicultura, Universidade Federal de Santa Catarina (UFSC), Rod. Admar
Gonzaga 1346, 88034-001 Florianpolis, SC, Brazil
b
Laboratrio de Camares Marinhos, UFSC, Beco dos Coroas 503, 88061-600 Florianpolis, SC, Brazil
c
Instituto Federal Catarinense (IFC), Campus Araquari., BR 280, Km 27, 89245-000, Araquari, SC, Brazil
d
Unidade de Reproduco 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 Agropecuria e Extenso Rural de Santa Catarina (EPAGRI), Rua Admar Gonzaga
1188, 88010-970 Florianpolis, SC, Brazil

a r t i c l e

i n f o

Article history:
Received 20 July 2015
Received in revised form 23 February 2016
Accepted 3 March 2016
Keywords:
Fish nutrition
Sorubim
Lactic acid bacteria
Weissela cibaria

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 catsh 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 mL1 in tubes containing de Man, Rogosa and Sharpe (MRS) medium. In particular,
Weissella cibaria (P36) reached 106 CFU mL1 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 prole of W. cibaria was determined molecularly by amplication of rRNA
16S gene was performed using PCR. Compared to control sh, W. cibaria-supplemented sh showed an
increase in RBC. These results show the efcacy 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/).

1. Introduction
Fish of the genus Pseudoplatystoma found in the Uruguay River,
Paraguay River and So Francisco River basins belong to the Pimelodidae family. They can reach 152 cm in length (Agostinho et al.,
2003) and are of great economic importance in Brazil (Roubach
et al., 2003). According to the Food and Agriculture Organization of
the United Nations (FAO, 2009), aquaculture production of South
American catsh species in continental waters in 2007 reached
about 670 tons, producing revenue close to $1,467,000.00 USD.

Corresponding author.
E-mail address: gabriel faj@hotmail.com (G.F.A. Jesus).

However, as reported by Moraes and Martins (2004), high production results in high stock density and equally high incidence of
disease. In particular, diseases of bacterial origin cause the greatest economic losses, often manifesting as subclinical changes in
infected sh (Martins et al., 2004). For example, the bacterial genus
Aeromonas caused primary and secondary septicemia in immunocompromised 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 disease in intensive sh farming. However, studies have reported an
increase in resistance to pathogens and growth in aquaculture
systems (Verschuere et al., 2000). Therefore, the use of probiotics is now considered a viable prophylactic alternative. Probiotics
are dened as live organisms that benet the health of the host

http://dx.doi.org/10.1016/j.aqrep.2016.03.001
2352-5134/ 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/).

J.L.P. Mouri
no et al. / Aquaculture Reports 3 (2016) 166171

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 22808 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
P19
P20
P21

bacillus
bacillus coccus
coccus
bacillus
bacillus
bacillus
coccus
coccus
coccus
coccus
Bacillus coccus
coccus
bacillus
coccus
coccus
coccus
coccus
coccus
coccus
coccus
coccus

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

0.00
10.00
0.00
10.00
9.33
10.50
0.00
0.00
5.33
0.00
8.33
0.00
10.67
12.33
0.00
9.33
8.33
0.00
0.00
3.00
8.67

P22
P23
P24
P25
P26
P27
P28
P29
P30
P31
P32
P33
P34
P35
P36
P37
P38
P39
P40
P41

coccus
coccus
coccus
coccus
coccus
bacillus coccus
bacillus coccus
coccus
bacillus coccus
bacillus coccus
coccus
bacillus
coccus
bacillus
bacillus
coccus
coccus
coccus
coccus
coccus

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

8.50
0.00
0.00
5.33
8.50
5.33
12.00
10.33
0.00
0.00
0.00
14.00
0.00
10.33
11.00
8.50
0.00
9.33
0.00
9.00

Determined by the diameter of the inhibition zone formed around the discs containing the probiotic bacterial strain using the WDA method.

Table 2
Antagonist activitya of lactic acid bacteria isolated from the intestine of hybrid surubim (Pseudoplatystoma reticulatum female x P. corruscans male) against Aeromonas
hydrophila subsp. hydrophila (AH), Aeromonas sobria (AS), Vibrio harveyi (VH), Vibrio alginolyticus (VA), Vibrio anguillarum (VAN), Enterococcus durans (ED), Pseudomonas
aeruginosa (PA), Escherichia coli (EC), Micrococcus luteus (ML), Staphylococcus aureus (SA), and Vibrio cholera (VC).
Inhibition zone (mm)
Strain
P2
P4
P6
P13
P14
P28
P29
P33
P35
P36
a

Species
N
Lactobacillus confusus
Lactobacillus brevis
Lactobacillus brevis
Lactococcus lactis lactis
N
Lactococcus lactis lactis
Lactobacillus brevis
Lactobacillus confusus
Weissella cibaria

AH
7.83
9.00
11.33
9.00
8.33
8.33
9.33
10.67
9.00
9.33

AS
6.00
8.00
8.83
8.00
6.00
6.00
6.00
6.00
6.00
6.00

PA
6.00
8.17
8.17
10.00
8.33
8.00
15.00
9.17
6.00
8.67

ED
6.00
6.00
6.00
6.00
6.00
6.00
6.00
8.33
6.00
6.00

VC
11.33
10.33
10.17
14.50
9.83
9.33
11.33
11.33
13.83
14.50

EC
6.00
10.00
10.33
10.33
8.33
6.00
8.33
10.33
9.17
11.83

ML
9.33
8.50
9.00
16.50
14.50
6.00
13.00
14.00
13.67
11.33

SA
6.00
9.00
10.00
8.17
8.00
6.00
8.33
7.67
6.00
8.67

VA
6.00
7.00
6.90
8.00
6.30
7.33
7.50
7.33
7.40
8.33

VAN
6.50
6.60
8.33
7.50
6.00
5.33
6.33
11.30
8.00
11.25

VH
5.50
7.40
8.20
6.50
6.00
6.00
6.00
8.33
9.20
10.00

Determined by the diameter of the inhibition zone formed around the discs containing the probiotic bacterial strains using the WDA method.

when administered in adequate concentrations (Reid, 2008). Such

benets include competitive exclusion of pathogenic bacteria, production of bacteriocins and organic acids, and immunomodulation
(De et al., 2014). Indeed, the use of probiotics has improved the
haemato-immunological parameters in sh (Barbosa et al., 2011;
et al., 2012; Ridha and Azad 2012). For example, using
Mourino
lactic acid bacteria isolated from the intestinal tract of Nile tilapia
(Oreochromis niloticus). Jatob et al. (2008) described improvement
in the immune system after experimental infection with Enterococcus durans.
This study aimed to investigate strains of bacteria isolated from
the intestinal tract of hybrid catsh (Pseudoplatystoma reticulatum
Linnaeus 1766 female P. corruscans Spix and Agassiz 1829 male)
to determine probiotic potential, ability to adhere to the intestinal epithelium, as well as modify host microbiota and improve
haematological parameters.
2. Materials and methods
2.1 Selection of probiotic strains
For lactic acid bacterial isolation, 20 healthy hybrid surubims
weighing 1.5 0.3 kg were obtained from Mar & Terra Ltda (Dourados, MS, Brazil). They were reared in tanks not previously treated
with chemicals or antibiotics. The animals were anaesthetized with
0.01% benzocaine. Their intestines were excised and then washed

Table 3
Incorporation of different lactic acid bacteria in the commercial diet and evaluation
of hydrogen ion dissociation (pH) in MRS media at 35 C for 48 h.
Strains

MRS media (CFU/mL)

pH

Diet (CFU/g)

Lactobacillus brevis (P6)

Lactobacillus brevis (P13)
Lactococcus lactis lactis (P29)
Lactobacillus brevis (P33)
Weissella cibaria (P36)

1.20 105
5.10 106
2.20 105
2.12 106
1.00 107

4.50
4.40
4.13
4.20
3.85

1.20 103
2.10 104
1.20 104
1.20 104
1.00 106

with sterile 0.65% saline solution to remove non adherent bacteria.

All animal procedures were approved by the Ethics Committee of
CEAU/UFSC (Protocol # 23080.021883/2009-91). The excised tissue was then homogenized in sterile 0.65% saline solution at a 1:1
ratio with the sample. The supernatant was removed and spread on
Man, Rogosa and Sharpe (MRS) Agar (HIMEDIA ) plates. The plates
were incubated at 35 C for 48 h. Colonies were again spread on
MRS plates for an additional 48 h to ensure purity and conrm the
characteristic morphology of Lactobacillus by Gram staining.
The following pathogenic bacterial strains were used for
in vitro functional antagonist assay: Aeromonas hydrophila subsp.
hydrophila (CPQBA 22808 DRM), Aeromonas sobria (isolated from
symptomatic sh), Vibrio harveyi (ATCC 14126), Vibrio alginolyticus (CPQBA 37812 DRM), Vibrio anguillarum (donated by Dr.
Marguerita Barraco UFSC), Enterococcus durans (ATCC 19492),

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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 g1 ).
Treatments

Totalheterotrophic

Lactic acid bacteria

Pseudomonas spp.

Enterococcus spp.

Unsupplemented sh
Supplemented sh

6.58 1.13a
5.71 0.14a

0.60 1.04a
3.72 0.98b

5.63 1.95a
4.76 0.72a

3.38 0.45a
2.27 0.07b

Different letters indicate statistical signicant difference between supplemented and unsupplemented sh by t-test (p < 0.05).

Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (D363),

Micrococcus luteus (A270), Staphylococcus aureus (ATCC 29213), and
Vibrio alginolyticus (isolated by Martins et al., 2010).
For bacterial inhibition, the strains noted above were reactivated, cultured, and maintained in Tryptic Soy Agar (TSA, Oxoid ) at
30 C for 24 h. For in vitro selection of antagonists, Pseudoplatystoma
species-specic pathogens were chosen, as well as other pathogens
of economic importance to aquaculture. The isolated strains were
rst evaluated for inhibition of A. hydrophila (CPQBA 22808 DRM)
according to the wet disc assay (WDA) described by Ramirz et al.
(2006). Isolates were inoculated in MRS agar plates at 108 CFU/mL,
as measured by spectrophotometry, and incubated at 35 C for 24 h.
After incubation, 1 cm discs were removed from the plates, and A.
hydrophila was inoculated on TSA agar plates. The discs from the
MRS plates were placed on top of the recently inoculated pathogen
agar and incubated at 30 C for 24 h. Antagonistic activity was measured by the diameter (mm) of the inhibition zone around the discs
placed in triplicate on each plate.
Ten strains with the greatest antagonistic capacity against A.
hydrophila were phenotypically characterized using the API 50CH
Kit (API, bioMrieux, Hazelwood, MO, USA). Lactic acid-consuming
bacteria were identied by the fermentation of 49 carbohydrates.
The results were interpreted according to APIWEB software (API,
bioMrieux, Hazelwood, MO, USA). Following evaluation of these
10 strains by WDA, only ve with the greatest antimicrobial potential were retained.
To interpret the inhibitory results, the pathogens were assigned
in different levels of importance: specic pathogens for aquaculture
(level 3); nonspecic pathogens for aquaculture in continental waters (level 2), and nonspecic pathogens for aquaculture
in marine waters (level 1). To assess the inhibition of marine
pathogens (Vibrio spp.) by WDA, TSA agar was enriched with 2%
NaCl. All bacterial cultures were routinely checked for purity by
Gram staining and depletion on plates.
Five selected pathogenic strains were then submitted to viability assays after inoculation in a commercial diet for carnivorous
sh with 45% crude protein. The ability to produce organic acids by
acidifying the culture medium was determined by the concentration of dissociated hydrogen ions (pH). The strains were tested to
determine colony forming units (CFU) per mL in tubes containing
MRS medium (pH 6) at 35 C for 48 h. After incubation, the concentration of dissociated hydrogen ions (pH) was measured for each
strain, assuming that the production of organic acids or hydrogen
peroxide caused any changes in the values (Vazquez et al., 2005).
Next, W. cibaria was added in a commercial diet consisting of the following recommended ingredients: calcium (max)
25.00 g kg1 , ether extract (min) 100.00 g kg1 , phosphorus (min)
10.00 g kg1 , brous material (max) 15.00 g kg1 , mineral material
(max) 100.00 g kg1 , crude protein (min) 400.00 g kg1 , and humidity (max) 120.00 g kg1 . A bacterial solution was then applied to the
diet as a spray at a ratio of 200 mL kg1 . After incorporation, the
diet was incubated with air circulation at 45 C for 18 h. Three 1 g
samples of supplemented dry diet were macerated in 1 mL of sterile
1.5% saline solution and serially diluted (1/10) nine times. Dilutions
of 105 and 109 were spread on MRS agar and incubated at 35 C
for 48 h to estimate a desirable concentration 106 CFU g1 . Control

Table 5
Haematological parameters of hybrid surubim (Pseudoplatystoma reticulatum female
x P. corruscans male) supplemented and unsupplemented with Weissella cibaria
(P36) in the diet at a concentration of 107 CFU.g1 . Red blood cells (RBC), white
blood cells (WBC), total thrombocytes count (TTC), haematocrit percentage (HTC),
basophils (BAS), eosinophils (EOS), granular leukocyte PAS+ (PAS), neutrophils
(NEU), lymphocytes (LIM), monocytes (MON), and glucose levels (GLC).
Parameters

Supplemented sh

Unsupplemented sh

RBC (x106 L1 )
TTC (x103 L1 )
WBC (x103 L1 )
HTC (%)
BAS (x103 L1 )
EOS (x103 L1 )
PAS (x103 L1 )
NEU (x103 L1 )
LIM (x103 L1 )
MON (x103 L1 )
GLC (mg dL1 )

1.85 1.6a
50.84 6.3 a
52.34 6.9 a
24 4.9 a
0
0.3 0.1 a
0.99 0.8 a
2.66 1.8 a
38.34 14.0 a
3.48 1.9 a
47.27 12.7 a

1.57 1.5 b
36.72 14.0 a
44.41 15.2 a
23.2 6.7 a
0
0.75 0.75 a
1.29 0.4 a
2.22 2.3 a
34.83 17.1 a
2.15 1.6 a
30.72 9.3 a

Different letters indicate statistical signicant difference between supplemented

and unsupplemented sh (p < 0.05).

diet was sprayed with the same concentration of sterilized MRS

medium, but without bacteria.
2.1. Intestinal tract colonization
Seventy-two healthy hybrids approximately 21 cm in length and
65 g in weight were obtained from Mar & Terra Ltda (Dourados,
Mato Grosso do Sul, Brazil). These sh were used to evaluate intestinal tract colonization by the W. cibaria selected in the previous step.
After 24 h starvation, the next steps included the following procedures. First, the intestinal tract was extracted with tweezers and
scalpel, washed twice with sterilized water, and homogenized in
a mortar with sterile saline solution serially diluted (1:10). Next,
the solution was spread on TSA agar for total heterotrophic bacterial count, MRS agar to determine lactic acid bacteria, Cetrimide
agar for Pseudomonas sp. selection, and TCBS agar for Vibrionaceae
selection. These were all incubated at 30 C. Total CFU counts were
determined after 24 h of incubation in TSA, Cetrimide, and TCBS
media and 48 h in MRS. The colonies grown on MRS agar were
Gram-stained and selected for subsequent reisolation and biochemical characterization (API test; bioMrieux) to conrm the
colonization and composition of host bacteria.
2.2. Experimental design
Twelve circular 100 L tanks with aeration and constant heating
(26 C) were used. The biological ltration system (gravel biolters) was independently maintained to ensure water quality (pH
6.87.2). Each day, sedimentary deposits and 70% of the water were
removed from the tanks. Water quality was measured as follows:
pH 6.8 0.2; total ammonia 1.2 0.4 mg/L; and dissolved oxygen
8.5 mg/L. Six sh were placed in each tank and acclimated for 5
days. After this period, they were fed with the experimental diets
twice a day for 15 days or unsupplemented diets (control). Fish
were randomly distributed in tanks divided into Weissela cibariasupplemented sh and control sh in 6 replicates.

J.L.P. Mouri
no et al. / Aquaculture Reports 3 (2016) 166171

2.3. Sampling
At the end of the experiment, the sh from each experimental
unit were sampled. Following anesthesia with eugenol (75 mg/L),
blood was collected from the caudal vessel using two 21 G 3 mL
syringes, one containing a drop of 10% EDTA as anticoagulant and
one without. The blood with anticoagulant was used for haematology analysis. The blood without anticoagulant was incubated for
2 h at 25 C, centrifuged at 1400g for 10 min to obtain the serum,
and stored at 20 C for glucose analysis.

98

2.5. Statistical analysis

Data were evaluated according to variance homogeneity by the
Bartlett test. In cases where variance heterogeneity was observed,
the data were transformed to log (x + 1) for both the microbiological
counts and blood counts and analyzed by t-test (p < 0.05).
2.6. Molecular identication
After the experiment, the bacteria were molecularly identied
at the State University of Campinas (UNICAMP). Using genomic
DNA extracted from the samples, rRNA 16S gene amplication was
performed with PCR. Primers used for PCR reaction were p27f and
p1401r homologous ends of the conserved 16S ribosomal RNA gene
of bacteria.
Amplication products were puried and sequenced using
an automated sequencer (MegaBACE 1000, GE Healthcare). The
primers used were p10f, 765f, 782r and p1100r. The partial
sequences of the 16S ribosomal RNA gene obtained from different primers were assembled into a contig and compared with
sequences of organisms found in the Genbank and RDP databases.
DNA sequences were aligned using the CLUSTAL X program
(Thompson et al., 1997), and phylogenetic analyses were conducted
using the MEGA 4.0 program (Tamura et al., 2007). Evolutionary distance matrix was calculated based on the model of Kimura (1980).
The phylogenetic tree was constructed from the evolutionary distances by the neighbor-joining method (Saitou and Nei, 1987) with
bootstrap values calculated from 1000 recounts using MEGA 4.0
software.
3. Results
3.1. Selection of probiotic strains
A total of 41 bacterial strains were isolated, all with morphological characteristics of Gram-positive lactic acid bacteria. Only 10
demonstrated inhibition zones > 10 mm, as determined by WDA
(Table 1). They were identied as Lactobacillus confusus (2 strains),
Lactobacillus brevis (3 strains), Lactococcus lactis lactis (2 strains),
Weissella cibaria (1 strain), and two unidentied strains by API 50CH
Kit (bioMrieux, Marcy lEtoile, France).
Strains that presented average inhibition zones >9 mm against
other pathogens were L. brevis (P6), L. brevis (P13), L. lactis lactis

Weissella koreensis S-5623 (AY035891)

Weissella kandleri DSM 20593 (M23038)

80

Weilssella soli DSM 14420 (AY028260)

56

Weissella confusa DSM 20196 (M23036)

46

98

Weiss ella cibaria CHJ3 (AF312874)

99

2.4. Haematology
Blood smears were stained by Giemsa/May Grunwald for differential count of leukocytes and total count of thrombocytes and
leukocytes (WBC) (Ishikawa et al., 2008). An aliquot was used for
hematocrit determination, and the remaining blood was stored
in ice to count the total number of erythrocytes (RBC) using a
Neubauer chamber (Ranzani-Paiva et al., 2013). An aliquot of serum
was used to determine glucose concentration by spectrophotometer (Spectrum SP-2100) at 505 nm according to Biotecnica Glucose
Kit (Biotecnica, Minas Gerais, Brazil).

169

COQBA 001-10 DRM 02

Weissella hellencia NCFB 2873 (X959810)
Weissella thailandensis FS61-1 (ABO23838)

99
71

Weissella parameseneroides DMS-20288 (M23033)

Weissella videscens DMS-20410 (M23040)

Fig. 1. Phylogenetic relationship among the sequences of 16S ribosomal RNA gene
of CPQBA 00110 DRM 02 and other microorganisms presented in the related
databases genbank and RDP.

(P29), L. brevis (P33), and W. cibaria (P36) (Table 2). These strains
reached a minimum concentration of 103 CFU.mL1 in the diet.
However, only W. cibaria (P36) reached 106 CFU.mL1 and was capable of reducing the pH of MRS to 3.85 (Table 3).
3.2. Intestinal tract colonization
Fish fed W. cibaria showed a higher value of total lactic acid
bacteria (3.72 104 CFU g1 ) in MRS compared to unsupplemented
sh (101 CFU g1 ). These results demonstrated that W. cibaria could
remain viable in the intestine of supplemented sh (Table 4). W.
cibaria still reduced the bacterial load of Streptococcus selectively
grown in KF agar. Supplemented sh showed a concentration of
102 CFU g1 bacteria; control sh presented 104 CFU g1 bacteria.
3.3. Haematology
Red blood cell count signicantly increased in sh supplemented with W. cibaria (p < 0.05). The other haemotological
parameters did not statistically differ (Table 5).
3.4. Molecular identication
The strain of W. cibaria was molecularly conrmed (Fig. 1) and
kept in the microorganism collection at the Multidisciplinary Center for Chemistry, Biology and Agriculture Research (CPQBA) of the
State University of Campinas (UNICAMP).
4. Discussion
This study investigated bacterial strains with probiotic potential
isolated from the mid-foregut of healthy hybrid surubim catsh (Pseudoplatystoma reticulatum female x P. corruscans male).
Forty-one strains of bacteria were isolated in this study. Seven
demonstrated inhibition zones >8 mm against pathogenic bacteria. Other studies have isolated bacterial strains with probiotic
potential. For example, Huys et al. (2001) and Fjellheim et al.
(2010) isolated 120 and 500 bacterial strains from Atlantic cod
(Gadus morhua) and turbot (Scophthalmus maximus), respectively.
Caipang et al. (2010) evaluated the in vitro antagonism of probiotic bacteria against common pathogens in the Atlantic cod.
They discovered dissimilarities in inhibition capacity among these
strains. Pan et al. (2008) also evaluated the in vitro antagonism of

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Clostridium butyricum CB2c, as well as its ability to adhere to intestinal 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, Jatob et al. (2008)
isolated L. plantarum from tilapia. Vazquez et al. (2005) isolated
Lactobacillus sp. from rainbow trout (Oncorhynchus mykiss). In the
present study, Weissella cibaria was able to reduce the pH of MRS
to 3.85. The mode of action of W. cibaria in reducing intestinal
pH in vivo and in vitro remains unknown. Nonetheless, this result
conrms the possibility of producing organic acids and hydrogen
peroxide to inhibit Gram-positive bacteria.
Apart from their inhibitory capabilities, the most important criterion for the selection and use of probiotic bacteria is colonization
in the intestinal tract (Verschuere et al., 2000). Specically, probiotic bacteria must be able to tolerate bile acids and adhere to
the intestinal epithelium (Joborn et al., 1997; Nikoskelainen et al.,
2001). In particular, isolated microbiota should colonize the digestive tract to a greater degree than commercial strains. In the present
study, W. cibaria reached values of 3.7 104 CFU g1 in the intestine
and lost only two orders of magnitude when analyzed in the diet.
Vendrell et al. (2008) counted 106 CFU g1 of probiotic in the diet.
Similar results were found by Jatob et al. (2008) for L. plantarum
colonizing the digestive tract of Nile tilapia (106 CFU mL1 ) and in
the diet (108 CFU mL1 ). However, in contrast to that reported by
Jatob et al. (2008), W. cibaria did not provoke a reduction in Pseudomonas concentration, but rather a decrease of Enterococcus spp.
concentration.
There is ample evidence that probiotic bacteria can reach
high concentrations in the intestinal tract, thus beneting animal
health (Gomez and Balcazar, 2008). In the present study, probioticsupplemented sh showed a signicant increase in RBC count
compared to control sh. This was also reported in Nile tilapia fed
L. plantarum (Jatob et al., 2008) and in Pseudoplatystoma sp. fed
et al., 2012).
with a symbiotic diet (inulin and W. cibaria) (Mourino
Increased values of RBC improve oxygen supply to sh tissues (De
Pedro et al., 2005), which could account for the more efcient circulatory supply in supplemented sh.
Very important consideration in probiotic selection is safety
prole. According to Nikoskelainen et al. (2001), members of the
Lactobacillus genus have been used in food products as probiotic
organisms and are usually considered safe. Indeed, previous studies
(Nikoskelainen et al., 2003) reported improved immunity conferred by L. rhamnosus JCM 1136. This result indicates that probiotic
supplementation can be an effective biotherapy or prophylactic
method in aquaculture. Perez-Sanchez et al. (2011) observed the
absence of furunculosis symptoms in rainbow trout fed with lactic
acid bacteria (L. lactis, L. plantarum, L. fermentum). These bacteria
have been characterized as probiotics able to inhibit the growth of
pathogenic bacteria. However, more studies should be conducted to
investigate the use of food additives relative to altering host intestinal microbiota and increasing lactic acid bacteria concentration in
the diet (Yousean and Amiri, 2009).
5. Conclusions
This study established the efcacy of isolating probiotic bacteria
from the intestine of the hybrid surubim and shows W. cibaria as a
promising bacterium for use in surubim aquaculture.
Acknowledgements
The authors thank the National Council for Scientic and Technological Development (CNPq/MAPA 577657/2008-9) for nancial
support and the grant to M.L. Martins (CNPq 302493/2010-7); Ministry of Fishing and Aquaculture of the Federal Republic of Brazil

Mar & Terra Ltda for sh donation; Drs. E. Zaniboni Filho, R.Y. Fujimoto, A.R.M. Magalhes, W.Q. Seiffert and R.P. Schocken-Iturrino
for critical review of the manuscript.
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