Aquaculture Reports 11 (2018) 59–69

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Aquaculture Reports
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Evaluation of probiotic potential of Bacillus spp. isolated from the digestive T

tract of freshwater fish Labeo calbasu (Hamilton, 1822)
⁎
Mathialagan Kavitha, Manickam Raja, Pachiappan Perumal
Department of Biotechnology, Periyar University, Periyar Palkalai Nagar, Salem 636 011, Tamil Nadu, India

A R T I C LE I N FO A B S T R A C T

Keywords: The probiotic use in aquaculture helps to improve the survival, growth and health of fish as it is a sustainable
Bacillus aquaculture practice. In the present study, Bacillus spp. were isolated from the digestive tract of freshwater fish,
Probiotic Labeo calbasu towards the assessment of their probiotic potential. Based on the preliminary screening on 64
Survival isolates obtained from the digestive tract of L. calbasu, only the Bacillus strains FS1, FC3 and FC6 were selected
Antibiotic resistance
by using Bacillus selective media (Himedia) for their probiotics characterization. The biosafety assay confirmed
Fish pathogens
Antagonistic activity
that the isolates were not pathogenic to the host fish. Further, the selected isolates Viz; FS1, FC3 and FC6 were
Labeo calbasu evaluated for probiotic properties. These strains were able to survive in acidic and alkaline conditions, higher
tolerance to bile salt, high surface hydrophobicity to solvents, and they were found to tolerate in gastric juice. All
the three isolates have exhibited notable amylase, proteolytic, lipase activity and susceptibility to various an-
tibiotics. Their antagonistic activity was assessed against the fish pathogens; Aeromonas hydrophila (KX756709),
A. veronii (KX688046), Acinetobacter junii (KX756708), A. tandoii (KX775222) Acinetobacter sp. (KX775221), and
Pseudomonas stutzeri (KX721473) that were isolated from the naturally infected fish, Dawkinsia filamentosa.
Among the three isolates, the FC6 exhibited a good antagonistic activity against three fish pathogens Viz;
Aeromonas hydrophila, Acinetobacter sp. and Acinetobacter tandoii; FS1 and FC3 strains did not show any activity
against the pathogens. Time killing kinetics assay also revealed that pathogens treated sample attained sta-
tionary phase quickly when compared to the control. Only the FC6 isolate had exhibited positive results for
biofilm formation assay. All the three isolates did not exhibit any haemolytic activity. The isolated species were
identified and confirmed on the basis of their colony morphology, biochemical characteristics and 16S rRNA
sequencing. The probiotic strains, FS1, FC3 and FC6 were identified to be Bacillus subtilis (KX756706), Bacillus
cereus (KX756707) and Bacillus amyloliquefaciens (KX775224) respectively. Based on the presently observed
characteristic features, it is concluded that the FC6 strain, Bacillus amyloliquefaciens found in the digestive tract
of L. calbasu could form a potential probiotic candidate.

1. Introduction
Aquaculture has emerged as a fastest growing industry as it offers
high-quality animal protein to augment nutritional and food security.
Globally, carp fishes form a most important group of aquaculture spe-
cies, as they contribute for over 72% of total freshwater fish production
(Swapna et al., 2010). In view of the majority of consumers preference
of Labeo calbasu coupled with its wide distribution in major riverine
systems and reservoirs in India, the Labeo fish is considered to be a most
essential carp species next only to the three other Indian major carps,
i.e., catla (Catla catla), rohu (Labeo rohita) and mrigal (Cirrhinus mrigala)
(Chondar, 1999). The major problem being encountered in aquaculture
industry is the outbreak of infectious - bacterial diseases, that results in
declined fish production (Wang et al., 2008). The disease outbreaks are
habitually caused by pathogenic microbes belonging to the genera;
Aeromonas, Vibrio, Edwardsiella, Pseudomonas and Streptococcus and one
of the major routes of infection is the mucosal surfaces of the digestive
tract of fish (Sugita et al., 1996; Defoirdt et al., 2011). To sort out these
problems, antibiotics have been widely used for many years against that
diseases and also growth promoters have been applied to improve the
animal performance in aquaculture (Done et al., 2015). However, the
continuous applications of antibiotics brings important changes in the
microbiota of the aquaculture systems, causing the development of
bacterial resistance to frequently used antimicrobials (Resende et al.,
2012) and which even affect the natural beneficial bacteria (He et al.,
2010, 2011, 2012). Hence, it is important to pursue and combat only

⁎
Corresponding author.
E-mail address: perumalarticles@gmail.com (P. Perumal).

https://doi.org/10.1016/j.aqrep.2018.07.001
Received 23 January 2018; Received in revised form 6 June 2018; Accepted 3 July 2018
Available online 17 July 2018
2352-5134/ © 2018 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/).
M. Kavitha et al.
the harmful pathogens by developing of alternative methods and the
use of probiotics would restrict the growth of pathogens (Gatesoupe,
1999).
Of late, the application of probiotics is considered to be an alter-
native measure for controlling the growth of pathogenic bacteria
without any side effects (Rinkinen et al., 2003; Magnadottir (2010) and
attempts have been made by researchers to isolate probiotic bacteria
from both the indigenous and exogenous microbiota of aquatic animals
(Reddy, 2015). The major probiotic mechanisms of action include the
enhanced epithelial barrier, increased adhesion to intestinal mucosa,
concomitant inhibition of pathogen adhesion, competitive exclusion of
pathogenic microorganisms and production of anti-microbial sub-
stances besides modulation of the immune system (Bermudez-Brito
et al., 2012). The acid and bile tolerance is an important parameter for
the ability of the probiotic microorganism to survive during the passage
through the upper gastrointestinal tract, particularly in an acidic con-
dition of the stomach (Hyronimus et al., 2000; Erkkila and Petaja,
2000). The tolerance to bile salts is essential for colonization and me-
tabolic activity of bacteria in the small intestine during the host
(Havenaar et al., 1992). In the intestine of their effective colonization,
the ability to adhere to the epithelial cells and mucosal surfaces has
been considered to be an important property of many bacterial strains
that are used as probiotics (FAO/WHO, 2001). Cell adhesion is a
complex process involving the interaction between the bacterial cell
membrane and interacting surfaces (Boonaert and Rouxhet, 2000).
The rich nutrients of the fish digestive tract provides conducive
environment for the bacterial growth (Kar et al., 2008). In the fish di-
gestive tract, the bacterial flora exhibit very broad and variable enzy-
matic potential as they play an important role in the fish digestive
process (Ray et al., 2010). The gastrointestinal bacterial flora of fishes is
capable of producing extracellular enzymes such as proteolytic, amy-
lolytic, cellulolytic, lipolytic, and chitinolytic enzymes, that are in-
volved in the digestion of proteins, carbohydrates, cellulose, lipids and
chitin in the host (Bairagi et al., 2002; Ray et al., 2012) and they also
promote the nutritional benefits in cultivable fish (Dutta et al., 2015).
Gastric juices are produced by the fish gut-walls which composed of
enzymes like pepsin that break down the proteins of food and hydro-
chloric acid. Before reaching the intestinal tract, the probiotic bacteria
must first transit through the stomach (Henriksson et al., 1999).
Therefore, tolerance to gastric juice constitutes the basic condition of
probiotic bacteria (Kobayashi et al., 1974). Some of the probiotic bac-
terial strains were able to pass through the stomach without undergoing
significant losses in their viability (Robins-Browne and Levine, 1981;
Havenaar et al., 1992). For the in vitro experiments, the acid tolerance
and artificial gastric juice tolerance test has been commonly followed to
select viable probiotic strains (Kim et al., 2006).
Biofilms consist of thickly packed microbial cells surrounded by the
self-synthesized extracellular polymeric matrix mainly comprising of
polysaccharides that are attached to a surface including biological tis-
sues (Stoodley et al., 2002). The Biofilms forming microbes have been
found to show high tolerance to exogenous stresses and the treatment
with conventional antibiotics are often ineffective in their ability to
eradicate microorganisms residing within the biofilm (Hall-Stoodley
and Stoodley, 2002; Rendueles et al., 2013). The haemolytic activity
assay is considered to be an important probiotic screening process.
Hemolysin is a very common virulence factor, which frequently causes
anemia and edema in the host, and hence, haemolytic strains are not
recommended for use as feed additives (Ouwehand et al., 2005).
Therefore, the non-haemolytic strains would be preferable for probiotic
use (Nandi et al., 2017a). The residual antimicrobials in aquatic pro-
ducts cause problems for the human health (Caruso et al., 2013). Cur-
rently, several probiotics and prebiotics are being developed to control
fish disease and to maintain a healthy microbial aquatic environment
(Verschuere et al., 2000).
Among the probiotics, the microbes of the genus, Bacillus have been
commonly used in humans and animals because of their ability to
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Aquaculture Reports 11 (2018) 59–69
produce antimicrobial substances coupled with their sporulation ca-
pacity, that confer them double advantage in terms of survival in dif-
ferent habitats (Abriouel et al., 2011). Since Bacillus bacteria secrete
many exoenzymes (Moriarty, 1996, 1998), many bacillus forms are
being commonly employed as putative probiotics (Rengpipat et al.,
1998, 2000; McIntosh et al., 2000; Oggioni et al., 2003). When com-
pared to the Lactobacillus spp., the Bacillus spp., exhibit important
probiotic features owing to their inherent potential to produce spores
which confers them heat-tolerance and longer shelf-life (Ramakrishnan
et al., 2008; Geng et al., 2012; Raida et al., 2003).
A number of gram negative bacteria belonging to the genera;
Aeromonas, Citrobacter, Pseudomonas, Proteus and some gram positive
bacteria of the genera, Streptococcus and Staphlycoccous cause serious
damages to the aquaculture industry (Welker et al., 2005; Verma et al.,
2006). Among these, Aeromonas hydrophila and A. salmonicida are
considered to be the most common fish pathogens that cause different
types of fish diseases like ulcer disease, carp erythrodermatitis and
motile Aeromonas septicemia (Lightner et al., 1992). Labeo rohita, L.
calbasu, Catla catla and Cirrhinus mirgala are the major cultivable species
in Indian aquaculture. These carps are being frequently affected by
bacterial infections that are associated with tail and fin rot, hemor-
rhagic septicemia and epizootic ulcerative syndrome (Vivas et al.,
2004). The bacterial infections decrease the yield to fish cultivators.
Hence, it is need the of the hour to find out and defend harmful pa-
thogens through alternative methods. As of now, we have only scarce
information on the probiotic properties of gut endosymbionts isolated
from freshwater fishes (Mukherjee and Ghosh, 2016; Muthukumar and
Kandeepan, 2015). The aim of our present investigation was to isolate,
identify and characterize the probiotic Bacillus spp. from the intestinal
microbiota of Labeo calbasu and to determine their safety and probiotic
characteristics as well as their in vitro ability to control the pathogenic
property against fish pathogens.
2. Materials and methods
2.1. Sample collection
Samples of Indian major carp, L. calbasu (Hamilton 1822) with an
average weight of 50 g were collected from the Stanley River, Mettur
Dam, Salem District, Tamil Nadu, India and transported alive to the
laboratory in polythene bags containing oxygenated water.
2.2. Isolation of gut bacteria
Under sterile conditions, the fish gut was dissected out and homo-
genized with 5 ml of normal saline. The homogenate was kept in a
boiling water bath at 80 °C for 20 min to remove fungal contaminants.
The homogenate was serially diluted and pour plated onto Bacillus agar
medium (Himedia- Mumbai, India). Plates were incubated under
aerobic conditions at 37 °C for 24 h. Individual colonies were picked up
and purified by streaking method in a fresh Bacillus agar medium. All
the isolates were identified and stored at −80 °C in nutrient broth
supplemented with 40% glycerol.
2.3. Pathogenic bacterial strains used
Six potent fish pathogenic bacterial strains were selected for the
present study Viz; Aeromonas veronii (KX688046), A. hydrophila
(KX756709), Pseudomonas stutzeri (KX721473), Acinetobacter sp.
(KX775221), Acinetobacter junii (KX756708) and Acinetobacter tandoii
(KX775222) from naturally infected fish Dawkinsia filamentosa that
were confirmed previously by Kavitha et al. (2017).
2.4. Screening of antimicrobial activity of the isolated strains
The Bacillus bacterial strains namely FS1, FC3 and FC6 were isolated
M. Kavitha et al.
from the digestive tract of L. calbasu were cultured in nutrient broth and
incubated for 14 h at 37 °C in continuous shaking mode. The potential
probiotic strains were tested for their antimicrobial activity by the cross
streak method (Monthon and Songtham, 2008) and agar well diffusion
method (Schillinger and Lucke, 1989) against the target fish pathogens.
2.5. Quantitative analysis of antimicrobial activity
Antimicrobial activity of the selected strains; FS1, FC3 and FC6 were
then determined quantitatively by time killing kinetics according to
Monthon and Songtham method (2008). In brief, 1 ml of freshly cul-
tured pathogenic bacterial strains were separately mixed with 100 ml of
sterile LB broth medium along with 100 μl culture supernatants of FS1,
FC3 and FC6 bacterial strains and were incubated at 37 °C in continuous
shaking mode. Optical Density (OD) was measured at 600 nm for 15 h
at 1 h interval. Culture medium of pathogenic bacterial strains without
culture supernatant of FS1, FC3 and FC6 was taken as control.
2.6. Bio-safety assay
The in vivo safety assessment is an important step before using the
selected probiotic candidates; FS1, FC3 and FC6 for commercial pur-
pose. Presently, healthy Labeo calbasu of the average weight of
50–100 g obtained from the Cauvery reservoir, Mettur Dam were di-
vided into four experimental groups (Three groups for treatment and
another group for control and each group containing 12 animals), each
with three replicates. The fishes were acclimatized in the laboratory
condition for 10 days. The selected probiotic candidates; FS1, FC3 and
FC6 were grown in nutrient broth (pH 7.2) at 37 °C for 24 h and cen-
trifuged (8000 rpm for 15 min) after the pellets were suspended in
sterile physiological saline (0.8% NaCl). Experimental fishes were in-
jected intraperitoneally with 100 μl of bacterial suspension (approxi-
mately 109 CFU/ml), whereas the control group was injected with
sterile saline (Mukherjee and Ghosh, 2016). Fishes in each group were
fed at 2% of the body weight per day for a period of 10 days. The
swimming behavior and health status were monitored every day for 10
days. After 10 days, both control and experimental fish were sacrificed
and examined the disease symptoms (Sharifuzzaman and Austin, 2009).
2.7. In-vitro tests for probiotic potential of Bacillus strains-determination of
optimal growth and pH
For the determination of optimal growth and pH of Bacillus species,
fresh overnight culture of Bacillus species were inoculated into LB broth
with varying pH (1–10). The pH were adjusted with concentrated acetic
acid (99%) and 5 N NaOH. The inoculated broth were incubated in
room temperature (37 °C) for 24 h. After 24 h of incubation, the growth
of the bacteria was measured using a spectrophometer, reading the
optical density at 600 nm (OD) against the uninoculated broth.
2.8. Bile tolerance
Crude bile was collected from the gall bladder of L. calbasu, using a
syringe and stored at –20 °C until use. Bile salt resistance of the three
isolates; FS1, FC3, and FC6 were determined by overnight cultures of
the bacterial strains that were inoculated in TSB broth containing 2.5,
5.0, 7.5, and 10% of bile salts followed by incubation at 37 °C for 3 and
6 h. The treated cells were then assessed by absorbance at 595 nm using
ELISA reader (Bio-Rad). Survivability of the isolates was represented by
percentage.
2.9. Cell surface hydrophobicity assay
Cell surface hydrophobicity of selected isolates was determined
according to the method of Thapa et al. (2004). The toluene, chloro-
form, and ethyl acetate, were used to detect the isolates surface
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Aquaculture Reports 11 (2018) 59–69
hydrophobicity. The overnight grown cells were collected by cen-
trifugation at 6000g, washed three times with PBS, resuspended in
10 ml Ringer’s solution, and OD at 600 nm was measured (A0) as
control. In tested sample, the cell suspension was mixed with equal
volume of solvent by vortexing for 2 min and kept at room temperature
for 30 min. The aqueous phase was removed and absorbance measured
at 600 nm (A1). The hydrophobicity of bacterial adhesion to solvent
was calculated using the formula: (1 − A1/A0) × 100. Percentage
values lower than 50% were considered as hydrophilic and values
higher than 50% were considered as hydrophobic (indicating the nature
of cell surface). The percent hydrophobicity was compared with that of
positive control. Among the selected isolates, those with comparatively
better survival capacity at digestive transit and hydrophobicity were
screened for their adhesion and invasion abilities.
2.10. Gastric juice tolerance
Gastric juice tolerance was evaluated by following the protocol
described by Ahire et al. (2011). Cell suspension was diluted to 1:10 in
synthetic gastric juice (pH 2.5) and incubated at 37 °C. The survival rate
of the isolates was measured at 0, 0.5 and 3 h by spreading on Bacillus
agar plates that were then incubated at 37 °C for 24 h. Tolerance rates of
the isolates in the presence of gastric juice were represented in CFU
ml−1 and percentage.
2.11. Quantitative enzyme activity of the selected bacterial strains
Extracellular amylase, protease, lipase and cellulase activities of the
selected bacterial strains were quantitatively estimated in starch-agar,
skim milk-agar, Tween20-agar and carboxymethylcellulose (CMC)-
agar media plates, respectively by following the methods of Kéléké
et al. (1995), Uyar et al. (2011), Denison and Koehn (1997) respec-
tively.
2.12. Antibiotic susceptibility assay
Antibiotic susceptibility of the selected three isolates was evaluated
against the selected antibiotics using disc diffusion method of
Thankappan et al. (2015). The antibiotics used in the susceptibility test
were; ampicillin, amoxicillin, penicillin, vancomycin, gentamycin, er-
ythromycin, chloramphenicol and ciprofloxacin. Antibiotic discs were
placed on to the Muller-Hindan agar plates and incubated at 37 °C for
24 h. Zone of inhibition was measured (mm), and the antibiotic sensi-
tivity was recorded based on their activity.
2.13. Detection of biofilm formation (Congo red agar method)
This method is based on the characteristic morphology of biofilm-
forming bacteria on Congo red medium. The isolates were streaked on
the Muller Hinton agar (HIMEDIA) supplemented with 0.8 g/l of Congo
red dye and incubated for 48 h at 37 °C. The production of black co-
lonies with a dry crystalline consistency indicated the biofilm formation
and non-biofilm- producing strains develop red colonies (Ramesh et al.,
2015).
2.14. Haemolytic activity
The selected isolates were further subjected to a haemolytic activity
by following the procedure described by Argyri et al. (2013). All the
isolates were streaked on blood agar plates containing 5% (w/v) human
blood and incubated for 48 h at 37 °C. The plates were examined for α-
haemolysis, β-haemolysis or non- haemolytic properties.
2.15. Characterization of the selected isolates
Identification was performed based on their morphological and
M. Kavitha et al.
biochemical characteristics Viz; Gram staining, Motility, Spore forma-
tion, Catalase, Oxidase, Urease, Nitrate to Nitrite, H2S Production,
Citrate, Indole test, Methyl red and Voges–Proskauer as described in
Bergey’s Manual of Systematic Bacteriology (Claus and Berkeley, 1986).
The carbohydrate fermentation tests with different carbohydrates were
performed according to manufactures instructions (HiCarbo™ Kit -Part
A, B & C).
2.16. DNA extraction, PCR amplification, identification and nucleotide
sequence accession number
The isolates were grown in nutrient broth in a rotary shaker at 37 °C
for overnight. The DNA was extracted using phenol-chloroform method.
The purity of DNA was checked by calculating the ratio of absorbance
readings at 260 nm and 280 nm. The values of 1.7–1.9 generally in-
dicate 85% purity. The 16S ribosomal RNA (rRNA) gene was amplified
using universal primer (Bioserve, India) 295367F (5′-GTGCTGCAGAG
AGTTTGATCCTGGCTCAG-3′), and 295268R (5′-CACGGATCCTACGGG
TACCTTGTTACGACTT-3′). The DNA products were amplified by PCR
using Applied Biosystems under the following conditions: initial dena-
turation 94 °C for 5 min, followed by 25 cycles each consisting of 94 °C
of 1 min, 55 °C for 1 min and 72 °C for 1 min 30 s, following a final
extension step at 72 °C for 7 min. The PCR products were analyzed by
agarose gel (1.2% w/v) electrophoresis. Amplicons were eluted and the
purified DNA products were sequenced at Eurofins Genomics,
Bangalore (India). The obtained nucleotide sequences were compared
with the available sequences of Bacillus species in the National Center
for Biotechnology Information (NCBI) database using Basic Local
Alignment Search Tool (BLAST). The NJ tree was constructed with
MEGA 7. All the sequences of Bacillus strains were deposited in NCBI-
Genbank.
2.17. Statistical analysis
All the experiments were performed in triplicates, and the results
were subjected to one-way analysis of variance (ANOVA). The sig-
nificance of the differences between treatments was compared by
Tukey’s tests (P < 0.05). Data were analyzed by SPSS for Windows
version 15.0 (SPSS) and PRISM 5 software (Graph Pad Software Inc.,
USA). The P values less than 0.05 were considered to be statistically
significant.
3. Results
3.1. Fish gut bacteria and their antimicrobial activity
A total of 64 bacterial strains were isolated from the digestive tract
of L. calbasu. Among them 3 Bacillus strains Viz; FS1, FC3 and FC6 were
evaluated for their antimicrobial activity against 6 pathogens. The re-
corded antimicrobial activities of the potential probiotic isolates in the
form of cell free supernatant against fish pathogens are shown in
Table 1. Among the three bacterial strains, only the FC6 showed po-
tential antagonistic activity against three Gram negative fish patho-
genic bacteria (Aeromonas hydrophila, Acinetobacter tandoii and Acine-
tobacter sp.). The FS1 and FC3 strains did not show any activity against
all the pathogenic strains in both cross streak and Agar well diffusion
methods (Fig. 1a & b).
3.2. Time killing kinetics assay
Antimicrobial activity was further confirmed quantitatively by time
killing kinetics assay. The probiotic strains; FS1, FC3 and FC6 were
treated with target pathogens. The results showed that, apart from these
three strains, FC6 treated pathogens Viz; A. hydrophila, Acinetobacter sp
and A. tandoii have attained the stationary phase quickly. The max-
imum inhibitory activity was found in the pathogen, A. hydrophila.The
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Aquaculture Reports 11 (2018) 59–69
Table 1
In vitro inhibitory activity of probiotic Bacillus spp., isolated from Labeo cal-
basu against fish pathogens (isolated from naturally infected fish, Dawkinsia
filamentosa).
Name of the fish pathogens Test probiotic organisms
FS1 FC3 FC6
Aeromonas hydrophila - - +++
Aeromonas veronii - - -
Acinetobacter junii - - -
Acinetobacter spp. - - ++
Acinetobacter tandoii - - +
Pseudomonas stutzeri - - -
+ zone of inhibition between 1 and 2 mm, ++ zone of inhibition between 2
and 4 mm, +++ zone of inhibition above 4 mm.
results showed that lag phase duration was extended in each case when
compared to the control one. Results also revealed that pathogens
treated sample attained stationary phase quickly (Fig. 2).
3.3. Bio-safety assay
In the in vivo biosafety assay, all the experimental and control
groups did not show any pathological symptoms (viz., lesions, hemor-
rhage, edema, loss of scale and mucus) or mortalities as recorded after
10 days (after IP injections of probiotics).
3.4. Preliminary selection of probiotic isolates using in vitro functional
characteristics - pH tolerance
The selected isolates gave the promising results to pH tolerance test
under the different culture conditions. During the experiment, a gradual
increase in the growth of the isolated Bacillus spp. was occurred within
the pH range of 1.0–7.0 (Fig. 3). When the pH range was increased from
7.0 to 10.0, the growth of the bacteria was gradually deceased.This
finding show that the isolated Bacillus species could able to survive in
extreme acidic as well as alkaline conditions. All the
Bacillus strains showed highly significant difference (P < 0.001) in
different pH.
3.5. Bile tolerance
In order to determine the survival ability of the bacterial strains
(FS1, FC3 & FC6) in the host gastrointestinal tract, the viability per-
centage of cells with and without bile salt treatments was carried out
for 3-h and 6-h exposure. All the tested isolates were able to survive at
the increasing concentrations of bile salt (Fig. 4a & b). The survivability
percentage of viable cells was significantly higher in FC6 (91.62, 86.41,
72.08 and 65.36%), FC3 (70.37, 68.07, 63.51, and 57.48%) and FS1
(63.04, 57.730, 53.40, and 47.64%) after 3 h exposure, respectively at
the increasing concentration of bile salt (1.0, 2.5, 5.0 and 7.5%). Si-
milarly, the viability percentage of cells significantly decreased in FC6
(48.00, 44.31, 38.12, and 36.66%), FC3 (31.20, 30.91, 29.70, and
27.70%) and FS1 (35.17, 29.82, 28.00, and 24.30%) after 6 h exposure,
respectively at the increasing concentration of bile salt (1.0, 2.5, 5.0
and 7.5%). Highly significant differences (P < 0.001) were observed
both in the FS1 and FC6, whereas, the FC3 showed only moderately
significant differences (P < 0.01) between bile concentrations at the
survival rates of 3 h and 6 h exposure.
3.6. Hydrophobicity assay
Microbial adhesion of the selected bacterial strains (FS1, FC3 & FC6)
to xylene, toluene, chloroform, and ethyl acetate was tested to assess
the adhesion ability of the bacterial cell surfaces and the results are
shown in Fig. 5. Among the three bacterial strains (FS1, FC3 & FC6), the
M. Kavitha et al.
Fig. 1. Screening of antimicrobial activity by cross streak and well diffusion method against six bacterial pathogenic strains.
1. Aeromonas hydrophila, 2. A. veronii, 3. Acinetobacter junii, 4. Acinetobacter sp., 5. A. tandoii, 6. Pseudomonas stutzeri.
adhesion capacity of FC6 was significantly higher than other strains.
The percentage of recorded hydrophobicity were: for FC6, 86.65 in
xylene, 69.14 in ethyl acetate, 65.71 in chloroform & 57.07 in toluene;
for FS1, 45.08 in chloroform, 39.91 in ethyl acetate, 29.79 in toluene &
25.42 in xylene and in case of FC3, 43.1 in toluene, 41.66 in xylene,
35.86 in chloroform & 28.89 in ethyl acetate. The result showed that
adhesion capacity of the selected bacterial strains was calculated in
terms of percentage. The hydrophobicity percentage (H%) of FS1 and
FC6 showed the significant difference (P < 0.05).
3.7. Qualitative gastric juice tolerance assay
The viability rates of the isolates in the presence of gastric juices at
different time periods are shown in Table 2. The FC6 and FC3 showed
highest tolerance rates of 68.51% and 56.87% respectively, when
compared to the FS1 (43.96%) after 3 h of incubation.
3.8. Quantitative enzyme assays
Extracellular enzyme production rates of the bacterial strains; FS1,
FC3 and FC6 are shown in Supp. Table 1. The amylase activity was
determined for all the three isolates. Only the FS1 and FC6 strains
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showed the positive results. Further, the lipase activity was found only
in FS1 and FC6.
3.9. Antibiotic susceptibility
The results of antibiotic sensitivity test of the selected bacterial
strains are shown in Table 3. All the three selected isolates were highly
susceptible (more than 10 mm of zone of inhibition) to antibiotics Viz;
chloramphenicol, ciprofloxacin, erythromycin and vancomycin. The
strains FS1 and FC6 were found to be susceptible to all antibiotics. The
strain FC3 was found to be resistance to ampicillin, amoxicillin and
Ciprofloxacin.
3.10. Biofilm formation assay
The biofilm formed by the isolates were screened and confirmed by
Congo red agar method. Only the FC6 showed black colonies with a dry
crystalline consistency confirming the biofilm formation (Supp. Fig. 1).
3.11. Haemolytic activity
The haemolytic reactions were recorded through the observations of
M. Kavitha et al.
Fig. 2. Time kinetics assay against four pathogenic bacterial strains. P1- Aeromonas hydrophila; P2- A. veronii; P3- Acinetobacter junii; P4- Acinetobacter sp.; P5- A.
tandoii and P6- Pseudomonas stutzeri.
a clear zone around the colonies (β -haemolysis), a partial hydrolysis
and greening zone (α -haemolysis) or no reaction (γ-haemolysis).The
isolated bacterial strains (FS1, FC3 and FC6) had no clear transparent or
greenish zone surrounding their colonies on the blood agar plates. The
haemolytic activity of the selected bacterial strains did not show any
haemolytic activity against human blood in the blood agar medium.
3.12. Biochemical characterization of Bacillus spp
The strains; FS1, FC3 and FC6 were found to be rod shaped, en-
dospore forming and Gram-positive bacteria. It was also found that all
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the bacterial strains could utilize citrate, catalase, esculin, glucose,
dextrose, sucrose, glycerol, salicin and malonate. All other compounds
such as urea hydrolysis, indole, malibiose, L-arabiose, dulcitol, arabitol,
α-methyl-D-glucose, cellobiose, melezitose, α- Methyl-D- mannoside,
xylitol and sorbose were not utilized by all the three bacteria (Table 4).
On the basis of morphological and biochemical characteristics, the
species of the selected bacterial strains (FS1, FC3 and FC6) were
identified namely as Bacillus subtilis, Bacillus cereus and Bacillus amylo-
liquefaciens.
M. Kavitha et al. Aquaculture Reports 11 (2018) 59–69

Fig. 5. Cell surface hydrophobicity of probiotic isolate’s against various sol-

vents. Value are represented as mean ± SD. P values for the probiotic isolates
were determined (FS1-(P < 0.05), FC3-(P > 0.05) & FC6-(P > 0.001)).
Fig. 3. Bacterial growth at different pH levels based on the OD values obtained.
Significant difference is indicated by different letters.
Values are presented as mean ± SD and different letters indicate significant
difference between groups (FS1, FC3 & FC6) respectively (P < 0.001).
Table 2
Gastric juice tolerance analysis of Bacillus isolates in terms of CFU ml−1. Each
3.13. Identification by 16S rRNA sequencing and phylogenetic analysis
value is the mean ± standard deviation of three separate experiments.

The Partial 16S rRNA sequence analysis of the isolates showed high Name of the Isolates Viability of Bacteria in CFU ml−1 % of survivability
sequence homology with Bacillus species (99%). The sequences were (×103) after 3 h

deposited in the Genbank database and the following accession num- 0h 0.5 h 3h
bers were obtained: Bacillus subtilis KX756706, B. cereus KX756707 and
B.amyloliquefaciens KX775224, respectively Supp. Table 2. The se- FS1 (KX756706) 0.9366 0.5661 0.3561 43.96
quences were used to construct a phylogenetic tree using the FC3 (KX756707) 2.5076 1.3523 1.1090 56.87
FC6 (KX775224) 0.6466 0.4533 0.37 68.51
Neighbour-Joining method and evolutionary history was inferred. The
optimal tree with the sum of branch length = 0.05203781 is shown in
(Supp. Fig. 2). The percentage of replicate trees in which the associated
Table 3
taxa clustered together in the bootstrap test (1000 replicates) are shown Antibiotic susceptibilities of Bacillus strains.
next to the branches. The tree has been drawn to scale, with branch
Antibiotics FS1 FC3 FC6
lengths in the same units as those of the evolutionary distances used to
infer the phylogenetic tree. The evolutionary distances were computed Amoxicillin (AMS) + – +
using the Maximum Composite Likelihood method and are in the units Ampicillin (A/S) + – +
of the number of base substitutions per site. The analysis involved 14 Ciprofloxacin (CIP) + – +
nucleotide sequences. Codon positions included were 1st + 2nd Chloramphenicol(C) + + +
Erythromycin (E) + + +
+3rd + Noncoding. All the positions containing gaps and missing data Gentamicin (G) + + +
were eliminated. There were a total of 700 positions in the final dataset Penicillin (P) + + +
(Supp. Fig. 2). Vancomycin (VA) + + +

4. Discussion
The aquaculture industry has faced many challenges during the past
decade with the occurrence of resistant pathogens coupled with lim-
itations in the consumption of drugs for disease treatment (Dawood and
Koshio, 2016; Hai, 2015). Fish gut constitutes diverse unexplored
–, resistant; +, susceptible.
microorganisms and dynamic ecosystem that plays an important role in
food digestion, nutrient absorption, survival besides general health of
host–aquatic animals (Nayak, 2010). Accordingly, the growth, devel-
opment, and innate immunity of the host-fish most probably depends
on these gut microbes. In order to exploit the beneficial aspects of these

Fig. 4. a. Probiotics isolates bile salt tolerance after 3 h at 37 °C (FS1(P < 0.001), FC3(P < 0.05) & FC6 (P < 0.001) respectively. b. Probiotics isolates bile salt
tolerance after 6 h at 37 °C (FS1(P < 0.05), FC3(P > 0.05) & FC6(P < 0.05) respectively. Values are presented as mean ± SD and in terms of percentage. The
different letters notations indicate significant difference.

65
M. Kavitha et al. Aquaculture Reports 11 (2018) 59–69

Table 4 against various Gram-positive and Gram-negative pathogenic bacteria.

Biochemical characterization of probiotic bacteria isolated from fish gut. A. hydrophila has been found to be an important fish pathogen that
Biochemical test Test probiotic organisms causes epizootic ulcerative syndrome, fin rot, tail rot and hemorrhagic
septicemia, in Indian major carps (Pridgeon, 2012; Kozińska et al.,
FS1 FC3 FC6 2002). Aquaculture is emerging as the fastest growing food-producing
industry globally because of the increasing demand for food fish-pro-
Gram staining + + +
Motility + + + tein. However, the intensive culture of food fish has led to outbreaks of
Spore forming + – + various bacterial diseases that resulted in billions of dollars of annual
Catalase + + + economic losses to the aquaculture industry (Pridgeon and Klesius,
Oxidase – + + 2011).
Urease – – –
Rajeswari et al. (2005) have isolated the A. hydrophila from the
Nitrate to Nitrite – – +
H2S Production – – + dropsy-infected common carps; Catla catla and Cirrhinus mrigala with
Citrate + + + the symptoms of distended abdomen, loose scales, deep ulcers on the
Indole test – – – dorsal surface and extensive haemorrhages on their ventral part. Saikot
Methyl red + + –
et al. (2013) have demonstrated the presence of Aeromonas on the
Voges–Proskauer + – +
Motile Aeromonas Septicemia (MAS) infected Nile Tilapia. Further-
Carbohydrate fermentation: more, Sebastiao et al. (2015) have identified the Aeromonas veronii, A.
Lactose – – +
Maltose + – +
hydrophila and Pseudomonas fulva from the infected tilapia fish (Or-
Mannitol + – + eochromis niloticus), tambaqui (Colossoma macropomum), carp (Cyprinus
Sucrose + + + carpio), cachara (Pseudoplatystoma reticulatum) and pacu (Piaractus me-
Glucose + + + sopotamicus) in Brazil by direct colony PCR and 16S rRNA gene se-
Xylose + – +
quencing. Eissa et al. (2010) have demonstrated that different Pseudo-
Fructose + – +
Dextrose + + + monas species could cause septicemia in O. niloticus and the pathogenic
Galactose – – + species included P. anguilli septica, P. putida and P. aeruginosa. Recently,
Raffinose – – + Kavitha et al. (2017)have isolated the Aeromonas veronii, A. hydrophila,
Trehalose + + + A. sp., A. junii A. tandoii and P. stutzeri from the naturally infected
Melibiose – – –
L-Arabiose – – –
Dawkinsia filamentosa (Blackspot barb). Sariati et al. (2015) had found
Mannose + – – that the P. stutzeri from naturally infected fishes, collected from dif-
Inulin + – + ferent localities in Indonesia. Abdelkarim et al. (2010) have reported
Sodium gluconate – + + that the pathogenic strains viz; Candida utilis, Pseudomonas stutzeri and
Glycerol + + +
Pasteurella haemolityca were observed from the Artemia cysts and
Salicin + + +
Dulcitol – – – aquatic environments. Similarly, Kozinska et al. (2014) had isolated the
Inositol – – + A. johnsonii (2 isolates), A. lwoffii (2 isolates), A. junii/johnsonii (1 iso-
Sorbitol + – – late), A. calcoaceticus (1 isolate), and Acinetobacter sp. (2 isolates) from
Adonitol – – + the diseased rainbow trout and common carp cultured in Poland.
Arabitol – – –
Erythritol – + –
In recent years, many reports have appeared on the antagonistic
α- Methyl- D- glucoside – – – property of Bacillus spp. against the aquatic pathogens (Kaynar and
Rhamnose – – + Beyatli, 2012). Ramesh et al. (2015) have demonstrated that Bacillus
Cellobiose – – – species isolated from the gut of healthy Labeo rohita had shown an-
Melezitose – – –
tagonistic activity against bacterial fish pathogens. Likewise, the pre-
α- Methyl- D- mannoside – – –
Xylitol – – – sently isolated strain, FC6 showed maximum antimicrobial activity
ONPG + – – against the three tested fish pathogens Viz; Aeromonas hydrophila
Esculin hydrolysis + + + (KX756709), Acinetobacter sp. (KX775221) and Acinetobacter tandoii
D- Arabinose – – – (KX775222) but no activity was observed against Aeromonas veronii,
Citrate utilization + + +
Acinetobacter junii and Pseudomonas stutzeri. Chen et al. (2016a) have
Malonate utilization + + +
Sorbose – – – found the potentiality of B. amyloliquefaciens in controlling the vibriosis
and other fish pathogens in turbot (Scophthalmus maxima) (Chen et al.,
2016b). Selim and Reda (2015) have described the B. amyloliquefaciens
probiotic microbes, it is most essential to isolate and characterize them. that enhanced immune status and disease resistance in Nile tilapia
Many commercial probiotics presently available in the market for the (Oreochromis niloticus). Similar findings have also been reported for
use in aquaculture are often relatively ineffective, as many of them are European eel (Anguilla anguilla) (Lu et al., 2011), catfish (Ictalurus
isolated from non-fish sources (Ghosh et al., 2007). Administration of punctatus) (Ran et al., 2012) and Indian major carp (Catla catla) (Das
probiotics in sufficient quantities improves the health of the host by et al., 2013).
controlling and stimulating the fish immune system (Verschuere et al., Ramesh et al. (2015) have described that the antimicrobial activity
2000). Hence, the application of probiotics rather than antibiotic of bacillus was further confirmed by using time killing kinetics assay.
treatment is a good approach to control a disease, and to improve the The time killing assay is required to quantitate pharmacodynamics of a
host health and immunity. The beneficial effect of probiotics has al- putative antibacterial agent by displayed levels of time dependent
ready been reported in freshwater fish like L. rohita (Giri et al., 2012; bacterial inhibition that were different among the tested bacteria and
Nayak et al., 2007), Oreochromis niloticus (Aly et al., 2008) and Epine- the concentrations, regardless of being gram-negative or gram-positive
phelus bruneus (Harikrishnan et al., 2010). Therefore, gastrointestinal (Sim et al., 2014; Schaper et al., 2005; Asoso et al., 2018). Ramesh et al.
tract of a healthy fish could be the best source for the isolation of po- (2015) have found that the probiotic strain, Bacillus subtilis LR1 exerted
tential probiotics, for the control of infectious diseases in fish. good inhibitory activity against the gram-negative fish pathogenic
Presently, 64 strains were isolated from the digestive tract of L. bacteria. During the present investigation, among the selected Bacillus
calbasu, among them three Bacillus strains (FS1, FC3 & FC6) were ten- strains (FS1, FC3 and FC6), the FC6 exhibited maximum inhibitory
tatively identified by using Bacillus selective medium. An earlier report activity against the fish pathogens.
showed that several species of Bacillus have the antimicrobial property Safety evaluation of the selected candidate should be non-

66
M. Kavitha et al.
pathogenic to the host, which is an important precondition towards
consideration of it as probiotic (Verschuere et al., 2000). Presently, the
in vivo trial was carried out for safety evaluation of selected Bacillus
isolates (FS1, FC3 & FC6) and it was found the absence of pathological
signs or motility on L. calbasu. The findings of the present investigation
strongly confirmed that the selected probiotic strains (FS1, FC3 & FC6)
could be considered as safe probiotics in aquaculture industries. In a
few previous reports, some of the bacterial species of the genus, Bacillus
Viz; Bacillus cereus and Bacillus subtilis were found to be pathogenic or
opportunistic pathogenic to the host fish (Austin et al., 1995; Ryan and
Ray, 2014; Midhun et al., 2017). Based on the recent investigations,
several authors have reported that the B. subtilis (Banerjee et al., 2017),
Bacillus sp. (Nandi et al., 2017a,b) and B. amyloliquefaciens (Nandi et al.,
2017b) are safe to be as probiotics for aquaculture application.
In the present study, all the Bacillus strains have showed viability at
the level of pH 1–10. Among the Bacillus strains, FC6 exhibited max-
imum cell viability when compared to the FS1 and FC3 strains under
the acidic condition. Our results suggested that FC6 can be used as
probiotic as it can survive even at low pH environment (Nikoskelainen
et al., 2001; Fjellheim et al., 2010). The ability of the isolate to survive
and grow in the high concentration of bile in the stomach passage time
and adherence on the fish gut epithelium is important characteristics to
be noted. Presently identified isolates were able to survive in wide
range of bile concentration of 5% and even higher up to 7.5%, as re-
ported earlier (Jini et al., 2011; Giri et al., 2012). Lee et al. (2017) have
stated that B. amyloliquefaciens LN was able to survive transit through
the stomach and the intestinal environment where it could work ef-
fectively. Our results suggest that FC6 can be used as probiotic as it
could survive under low pH conditions and high bile salt concentra-
tions.
Adhesion to epithelial cells is a vital parameter to be considered to
be a potent probiotic, since it provides the ability to resist the instability
of the intestinal content (Guo et al., 2010). Colonization in the in-
testinal epithelial cell wall and mucosal surfaces is an important de-
sirable property of probiotic bacteria as it prevents the pathogenic
bacteria adhesion besides invading all the available space of the in-
testine and also it prevents the inflammatory reactions. The surface
properties like hydrophobicity demonstrated by the isolates could have
contributed to its adhesion property (Kos et al., 2003). Presently, the
highest adhesion capacity was observed from FC6 (86.65% in xylene,
69.14% in ethyl acetate, 65.71% in chloroform and 57.07% in toluene)
and followed by FS1 (45.08% in chloroform, 39.91% in ethyl acetate,
29.79% in toluene and 25.42% in xylene) and FC3 (43.1% in toluene,
41.66% in xylene, 35.86% in chloroform and 28.89% in ethyl acetate),
respectively. The adhesion capacity of the Bacillus spp. was much
higher when compared to the previous report (Thankappan et al.,
2015). These results indicated that FC6 might be able to survive transit
through the stomach, as compared to the other species and survive in
the intestinal environment where it could work effectively. In contrast,
some Bacillus spp. spores were stated to be susceptible to such condi-
tions (Smith, 1989).
Microbial population is one of the most important components of
the gut ecosystem which is essential for animal health and nutrition
(Ray et al., 2012). The present study confirms the extracellular enzymes
(amylase, protease and lipase) producing ability of selected bacterial
strains namely FS1, FC3 and FC6. According to Ray et al. (2012), the
extracellular enzyme producing bacteria in fish gut exercise positive
effects to the digestive processes of the host. Bacterial enzymes can be
helpful for digesting carbohydrates, proteins and specially substrate like
cellulose, which only few a animals can digest (Smith, 1989). There-
fore, application of probiotics with enzyme producing ability is gaining
attention in aquaculture so as to promote nutritional benefits to culti-
vable fish (Dutta et al., 2015). There are a number of published reports
on the ability of Bacillus spp. to produce extracellular enzymes Viz:
amylolytic, proteolytic, cellulolytic and lipolytic in the GI tracts of
tropical freshwater fish (Ray et al., 2012; Bairagi et al., 2002; Banerjee
67
Aquaculture Reports 11 (2018) 59–69
et al., 2013; Kar and Ghosh, 2008; Mondal et al., 2010). It is interesting
that presently, except FC3 all other isolates were found to be susceptible
to the antibiotics Viz; ampicillin, amoxicillin, streptomycin, penicillin-
G, gentamycin, erythromycin, chloramphenicol, kanamycin, tetra-
cycline, and rifampicin. Similar findings were earlier reported by
Thankappan et al. (2015) who have found susceptibility by the Bacillus
spp to the antibiotics Viz;ampicillin, amoxicillin, cephalexin, strepto-
mycin, penicillin-G, gentamycin, erythromycin, chloramphenicol, ka-
namycin, tetracycline, and rifampicin.
Moreover, the isolates that have antipathogenic property might also
have the ability to survive and colonize the fish digestive tract. And
also, the ability of bacteria to form biofilms helps to survive in hostile
environments within the host and is considered to be responsible for
chronic or persistent infections (Christensen, 1989). During the biofilm
formation assay by Congo red agar (CRA) method, out of three Bacillus
strains, only the FC6 displayed black colonies with dry crystalline
morphology suggesting that CRA method is suitable for screening of
biofilm forming bacteria (Zuberi and Nadeem, 2017).
Hemolysis is one of the pathogenic properties of bacteria, as it fa-
cilitates infection by microbial entry in to the small lesions in the skin
and mucous (Madigan et al., 1984). The present study confirmed that
the isolated Bacillus strains did not show any haemolytic activity and
hence it can be used with food ingredients for better health. Similarly,
Ramesh et al. (2015) have confirmed that Bacillus spp., have showed
the non-haemolytic activity.
The presently isolated microbes were gram positive, catalase ne-
gative that showed the ability to ferment esculin, glucose, dextrose,
sucrose, glycerol, salicin and malonate. Our biochemical results have
been found to be similar to the findings of earlier researchers (Chu
et al., 2010; Rajashekhar et al., 2017; Lee et al., 2017). As such, an
extensive research on Bacillus was initiated only 15 years ago (Hong
et al., 2005; Mazza, 1994; Sanders et al., 2003) and consequently a
number of Bacillus spp. were evaluated for their efficiency in various
livestock production sectors like cattle, poultry, and fishery. Presently
the isolated bacterial strains namely; FS1, FC3 and FC6 were identified
as Bacillus subtilis, Bacillus cereus and Bacillus amyloliquefaciens, re-
spectively by using morphological and biochemical tests and their
identification was subsequently confirmed through 16S rRNA gene se-
quencing (Shah et al., 2010).
5. Conclusion
The study shows that, the selected strain Bacillus amyloliquefaciens
FC6 could form a promising probiotic for controlling A. hydrophila in L.
calbasu. Further, in vivo evaluation studies are required to determine its
applications in aquaculture environment. The findings of the present
study support the strain selection and evidence for using probiotics as a
useful approach in enhancing resistance to infections.
Conflict of interest
All authors declare that they have no conflict of interest.
Acknowledgements
The authors thank the authorities of Periyar University, for pro-
viding the necessary facilities to carry out this work. The second author
(MR) is grateful to the UGC- New Delhi for the award of Dr. DSK
Postdoctoral Fellowship (Vide File No.F.42/2006 (BSR)/BL/1516/
0408).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.aqrep.2018.07.001.
M. Kavitha et al.
References
Abdelkarim, M., Kamel, C., Fathi, K., Amina, B., 2010. Use of Pseudomonas stutzeri and
Candida utilis in the improvement of the conditions of artemia culture and protection
against pathogens. Braz. J. Microbiol. 41, 107–115.
Abriouel, H., Franz, C.M.A.P., Omar, N.B., Galvez, A., 2011. Diversity and applications of
Bacillus bacteriocins. FEMS Microbiol. Rev. 35, 201–232.
Ahire, J.J., Patil, K.P., Chaudhari, B.L., Chincholkar, S.B., 2011. Food Chem. 127 (2),
387–393.
Aly, S.M., Ahmed, Y.A., Ghareeb, A.A., Mohamed, M.F., 2008. Studies on Bacillus subtilis
and Lactobacillus acidophilus, as potential probiotics, on the immune response and
resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections. Fish
Shellfish Immunol. 25, 128–136.
Argyri, A.A., Zoumpopoulou, G., Kimon, A.G., Karatzas, K.A., Tsakalidou, E., Nychas,
G.J., Panagou, E., Tassou, C., 2013. Selection of potential probiotic lactic acid bac-
teria from fermented olives by in vitro tests. Food Microbiol. 33, 282–291.
Asoso, O.S., Oladunmoye, K.O., Ogundare Proximate, A.O., 2018. Analysis and time
killing kinetics of Calotropis procera extracts on some selected pathogens. EJMP 22
(2), 1–8.
Austin, B., Stuckey, L., Robertson, P., Effendi, I., Griffith, D., 1995. A probiotic strain of
Vibrio alginolyticus effective in reducing diseases caused by Aeromonas salmonicida,
Vibrio anguillarum and Vibrio ordalii. J. Fish Dis. 18, 93–96.
Bairagi, A., Ghosh, K., Sen, S.K., Ray, A.K., 2002. Enzyme producing bacterial flora iso-
lated from fish digestive tracts. Aquacult. Int. 10, 109–121.
Banerjee, G., Ray, A.K., Askarian, F., Ringø, E., 2013. Characterization and identification
of enzyme-producing autochthonous bacteria from the gastrointestinal tract of two
Indian air-breathing fish. Benef. Microbes 4, 277–284.
Banerjee, G., Nandi, A., Ray, A.K., 2017. Assessment of hemolytic activity, enzyme pro-
duction and bacteriocin characterization of Bacillus subtilis LR1 isolated from the
gastrointestinal tract of fish. Arch. Microbiol. 199, 115–124.
Bermudez-Brito, M., Julio Plaza, D., Muñoz-Quezada, S., Carolina Gómez, L., Angel, G.,
2012. Probiotic mechanisms of action. Ann. Nutr. Metab. 61, 160–174.
Boonaert, C.J.P., Rouxhet, P.G., 2000. Surface of lactic acid bacteria: relationships be-
tween chemical composition and physicochemical properties. Appl. Environ.
Microbiol. 66, 2548–2554.
Caruso, D., Lusiastuti, A.M., Slembrouck, J., Komarudin, O., Legendre, M., 2013.
Traditional pharmacopeia in small scale freshwater fish farms in west java, Indonesia:
an ethno veterinary approach. Aquaculture 416, 334–345.
Chen, Y., Li, J., Xiao, P., Li, G.Y., Yue, S., Huang, J., Zhu, W.Y., Mo, Z.L., 2016a. Isolation
and characterization of Bacillus spp. M001 for potential application in turbot
(Scophthalmus maximus L) against Vibrio anguillarum. Aquacult. Nutr. 22, 374–381.
Chen, Y., Li, J., Xiao, P., Zhu, W., Mo, Z.L., 2016b. The ability of marine Bacillus spp.
isolated from fish gastrointestinal tract and culture pond sediment to inhibit growth
of aquatic pathogenic bacteria. Iran. J. Fish. Sci. 15, 701–714.
Chondar, S.L., 1999. Biology of Finfish and Shellfish. SCSC Publishers (India), Howrah,
West Bengal, India, pp. 1–514.
Christensen, B.E., 1989. The role of extracellular polysaccharides in biofilms. J.
Biotechnol. 10, 181–202.
Chu, W., Lu, F., Zhu, W., Kang, C., 2010. Isolation and characterization of new potential
probiotic bacteria based on quorum-sensing system. J. Appl. Microbiol. 110,
202–208.
Claus, D., Berkeley, R.C.W., 1986. Genus Bacillus Cohn 1872, 174AL. In: Sneath, P.H.A.,
Mair, N.S., Sharpe, M.E., Holt, J.G. (Eds.), Bergey’s Manual of Systematic
Bacteriology. Williams and Wilkins, Baltimore, USA, pp. 1105–1138.
Das, A., Nakhro, K., Chowdhury, S., Kamilya, D., 2013. Effects of potential probiotic
Bacillus amyloliquifaciens FPTB16 on systemic and cutaneous mucosal immune re-
sponses and disease resistance of catla (Catla Catla). Fish Shellfish Immunol. 35,
1547–1553.
Dawood, M.A., Koshio, S., 2016. Recent advances in the role of probiotics and prebiotics
in carp aquaculture: a review. Aquaculture 454, 243–251.
Defoirdt, T., Sorgeloos, P., Bossier, P., 2011. Alternatives to antibiotics for the control of
bacterial disease in aquaculture. Curr. Opin. Microbiol. 14, 251–258.
Denison, D.A., Koehn, R.D., 1997. Cellulase activity of Poronia oedipus. Mycologia 69,
592–601.
Done, H.Y., Venkatesan, A.K., Halden, R.U., 2015. Does the recent growth of aquaculture
create antibiotic resistance threats different from those associated with land animal
production in agriculture? AAPS J. 17, 513–524.
Dutta, D., Banerjee, S., Mukherjee, A., Ghosh, K., 2015. Selection and probiotic char-
acterization of exoenzyme-producing bacteria isolated from the gut of Catla Catla
(Actinopterygii: Cypriniformes: Cyprinidae). Acta Ichthyol. Piscat. 45, 373–384.
Eissa, N.M.E., AbouEl-Ghiet, E.N., Shaheen, A.A., Abbass, A., 2010. Characterization of
Pseudomonas species isolated from Tilapia Oreochromis niloticus in Qaroun and Wadi-
El-Rayan Lakes, Egypt. Global Vet. 5 (2), 116–121.
Erkkila, S., Petaja, E., 2000. Screening of commercial meat starter cultures at low pH in
the presence of bile salts for potential probiotic use. Meat Sci. 55, 297–300.
FAO/WHO, 2001. Evaluation of health and nutritional properties of powder milk and live
lactic acid bacteria. Rome, Italy: Food and Agriculture Organization of the United
Nations and World Health Organization Expert Consultation Report.
Fjellheim, A.J., Klinkenberg, G., Skjermo, J., Aasen, I.A., Vadstein, O., 2010. Selection of
candidate probionts by two different screening strategies from Atlantic cod (Gadus
morhua L.) larvae. Vet. Microbiol. 144, 153–159.
Gatesoupe, F., 1999. The use of probiotics in aquaculture. Aquaculture 180, 147–165.
Geng, X., Dong, X.H., Tan, B.P., Yang, Q.H., Chi, S.Y., Liu, H.Y., 2012. Effects of dietary
probiotic on the growth performance, non-specific immunity and disease resistance
of cobia, Rachycentron canadum. Aquacult. Nutr. 18, 46–55.
68
Aquaculture Reports 11 (2018) 59–69
Ghosh, S., Sinha, A., Sahu, C., 2007. Isolation of putative probionts from the intestines of
Indian major carps. Isr. J. Aquacult. 59, 127–132.
Giri, S.S., Sukumaran, V., Dangi, N.K., 2012. Characteristics of bacterial isolates from the
gut of freshwater fish, Labeo rohita that may be useful as potential probiotic bacteria.
Probiotics Antimicrob. Proteins 4, 238–242.
Guo, X.H., Kim, J.M., Nam, H.M., Park, S.Y., Kim, J.M., 2010. Anaerobe 16 (4), 321–326.
Hai, N.V., 2015. Research findings from the use of probiotics in tilapia aquaculture: a
review. Fish Shellfish Immunol. 45, 592–597.
Hall-Stoodley, L., Stoodley, P., 2002. Developmental regulation of microbial biofilms.
Curr. Opin. Biotechnol. 13, 228–233.
Harikrishnan, R., Balasundaram, C., Heo, M.S., 2010. Lactobacillus BK sakei 19 enriched
diet enhances the immunity status and disease resistance to streptococcosis infection
in kelp grouper, Epinephelus bruneus. Fish Shellfish Immunol. 29, 1037–1043.
Havenaar, R., Brink, B.T., Huis isn’t Veld, H.J.H., 1992. Selection of strains for probiotic
use. In: Fuller, R. (Ed.), In Probiotics. Chapman & Hall, London, pp. 209–224.
He, S., Zhou, Z., Liu, Y., Cao, Y., Meng, K., Shi, P., Yao, B., Ringø, E., 2010. Effects of the
antibiotic growth promoter’s flavomycin and florfenicol on the autochthonous in-
testinal of hybrid tilapia (Oreochromis niloticus×O. aureus). Arch. Microbiol. 192,
985–994.
He, S., Zhou, Z., Meng, K., Zhao, H., Yao, B., Ringø, E., Yoon, I., 2011. Effects of dietary
antibiotic growth promoter and Saccharomyces cerevisiae fermentation product on
production, intestinal bacterial community, and nonspecific immunity of hybrid ti-
lapia (Oreochromisniloticus female×Oreochromis aureus male). J. Anim. Sci. 89,
84–92.
He, S., Zhou, Z., Liu, Y., Cao, Y., Meng, K., Shi, P., Yao, B., Ringø, E., 2012. Do dietary
betaine and the antibiotic florfenicol influence the intestinal autochthonous bacterial
community in hybrid tilapia (Oreochromis niloticus×O. aureus)? World J. Microbiol.
Biotechnol. 28, 785–791.
Henriksson, A., Khaled, A.K.D., Conway, P.L., 1999. Lactobacillus colonization of the
gastrointestinal tract of mice after removal of the nonsecreting stomach region.
Microb. Ecol. Health Dis. 11, 96–99.
Hong, H.A., Duc, L.H., Cutting, S.M., 2005. FEMS Microbiol. Rev. 29 (4), 813–835.
Hyronimus, B., Le. Marrec, C., Hadi Sassi, A., Deschamps, A., 2000. Acid and bile toler-
ance of spore-forming lactic acid bacteria. Int. J. Food Microbiol. 61, 193–197.
Jini, R., Swapna, H.C., Rai, A.K., Vrinda, R., Prakash, M.H., Sachindra, N.M., 2011.
Isolation and characterization of potential lactic acid bacteria (LAB) from freshwater
fish processing wastes for application in fermentative utilization of fish processing
waste. Braz. J. Microbiol. 42, 1516–1525.
Kar, N., Ghosh, K., 2008. Enzyme producing bacteria in the gastrointestinal tracts of Labeo
rohita (Hamilton) and Channa punctatus (Bloch). Turk. J. Fish Aquat. Sci. 8, 115–120.
Kar, N., Roy, R.N., Sen, S.K., Ghosh, K., 2008. Isolation and characterization of extra-
cellular enzyme-producing bacilli in the digestive tracts of rohu, Labeo rohita
(Hamilton) and murrel, Channa punctatus (Bloch). Asian Fish. Sci. 21 (4), 421–434.
Kavitha, M., Raja, M., Kamaraj, C., Karthik, R.R., Balasubramaniam, V., Balasubramani,
G., Perumal, P., 2017. In vitro antimicrobial activity of Azadirachta indica (Leaves)
against fish pathogenic bacteria isolated from naturally infected Dawkinsia fila-
mentosa (Blackspot barb). Med. Aromat. Plants (Los Angels) 6, 294.
Kaynar, P., Beyatli, Y., 2012. Antagonistic activities of Bacillus spp. strains isolated from
the fishes. J. Appl. Biol. Sci. 6 (3), 77–81.
Kéléké, S., Kimpolo-Kimfoko, L., Brauman, A., 1995. In: Agbor Egbe, T.S., Brauman Alain,
T., Griffon, D. (Eds.), Transformation Alimentaire Du Manioc. Colloques et
Séminaires, Paris, pp. 319–330.
Kim, H.J., Shin, H.S., Ha, W.K., Yang, H.J., Lee, S.W., 2006. Characterization of lactic
bacterial strains isolated from raw milk. Asian-Aust. J. Anim. Sci. 19 (1), 131–136.
Kobayashi, Y., Toyama, K., Terashima, T., 1974. Biological characteristics of Lactobacillus.
II. Tolerance of a multiple antibiotic resistant strain, Lactobacillus casei PSR 3002, to
artificial digestive fluids. Nippon Saikingaku Zasshi 29, 691–697.
Kos, B., Susković, J., Vuković, S., Simpraga, M., Frece, J., Matosić, S., 2003. J. Appl.
Microbiol. 94 (6), 981–987.
Kozińska, A., Figueras, M.J., Chacon, M.R., Soler, L., 2002. J. Appl. Microbiol. 93 (6),
1034–1041.
Kozinska, A., Paździor, E., Pękala, A., Niemczuk, W., 2014. Acinetobacter johnsonii and
Acinetobacter lwoffii - the emerging fish pathogens. Bull. Vet. Inst. Pulawy 58,
193–199.
Lee, A., Cheng, K.C., Liu, J.R., 2017. Isolation and characterization of a Bacillus amylo-
liquefaciens strain with zearalenone removal ability and its probiotic potential. PLoS
One 12 (8), e0182220.
Lightner, D.V., Bell, T.A., Redman, R.M., Mohney, L.L., Natividad, J.M., Rukyani, A.,
Poernomo, A., 1992. A review of some major diseases of economic significance in
penaeid prawns/shrimps of the Americas and Indopacific. In: Shariff, I.M.,
Subasinghe, R.P.A. (Eds.), Diseases in Asian Aquaculture I. Fish Health Section. Asian
Fisheries Society, Manila, pp. 57–80.
Lu, L., Cao, H., He, S., Wei, R., Diong, M., 2011. Bacillus amyloliquefaciens G1: a potential
antagonistic bacterium against eel-pathogenic Aeromonas hydrophila. Evid.-Based
Complement. Altern. Med. https://doi.org/10.1155/2011/824104.
Madigan, M., Martinko, J., Parker, J., 1984. Brock Biology of Microorganisms, 9th ed.
Prentice Hall, Englewood Cliffs, NJ: USA.
Magnadottir, B., 2010. Immunological control of fish diseases. Mar. Biotechnol. (NY) 12,
361–379.
Mazza, P., 1994. Boll. Chim. Farm. 133 (1), 3–18.
McIntosh, D., Samocha, T.M., Jones, E.R., Lawrence, A.L., McKee, D.A., Horowitz, S.,
Horowitz, A., 2000. The effect of a commercial bacterial supplement on the high-
density culturing Litopenaeus vannamei with a low-protein diet in an outdoor tank
system and no water exchange. Aquacult. Eng. 21, 215–227.
Midhun, S.J., Neethu, S., Vysakh, A., Sunil, M.A., Radhakrishnan, E.K., Jyothis, M., 2017.
Antibacterial activity of autochthonous bacteria isolated from Anabas testudineus
M. Kavitha et al.
(Bloch, 1792) and it's in vitro probiotic characterization. Microb. Pathog. 113,
312–320.
Mondal, S., Roy, T., Ray, A.K., 2010. Characterization and identification of en-
zyme‐producing bacteria isolated from the digestive tract of Labeo bata. J. World
Aquacult. Soc. 41, 369–377.
Monthon, L., Songtham, S.A., 2008. Comparison of two methods used for measuring the
Antagonistic activity of Bacillus species. Walailak J. Sci. Technol. 5 (2), 161–171.
Moriarty, D.J.W., 1996. Microbial biotechnology: a key ingredient for sustainable aqua-
culture. Infofish Int. 4, 29–33.
Moriarty, D.J.W., 1998. Control of luminous Vibrio species in penaeid aquaculture ponds.
Aquaculture 164, 351–358.
Mukherjee, A., Ghosh, K., 2016. Antagonism against fish pathogens by cellular compo-
nents and verification of probiotic properties in autochthonous bacteria isolated from
the gut of an Indian major carp, Catla Catla (Hamilton). Aquacult. Res. 47,
2243–2255.
Muthukumar, P., Kandeepan, C., 2015. Isolation, identification and characterization of
probiotic organisms from intestine of fresh water fishes. Int. J. Curr. Microbiol. Appl.
Sci. 4, 607–616.
Nandi, A., Dan, S.K., Banerjee, G., Ghosh, P., Ghosh, K., Ringø, E., Ray, A.K., 2017a.
Probiotic potential of Autochthonous bacteria isolated from the gastrointestinal tract
of four freshwater teleost’s. Probiotics Antimicrob. Proteins 9, 12–21.
Nandi, A., Banerjee, G., Dan, S.K., Ghosh, P., Ghosh, K., Ray, A.K., 2017b. Screening of
autochthonous intestinal microbiota as candidate probiotics isolated from four
freshwater teleosts. Curr. Sci. 113 (4), 25.
Nayak, S.K., 2010. Probiotics and immunity: a fish perspective. Fish Shellfish Immunol.
29 (1), 2–14.
Nayak, S.K., Swain, P., Mukherjee, S.C., 2007. Effect of dietary supplementation of pro-
biotic and vitamin C on the immune response of Indian major carp, Labeo rohita
(Ham). Fish Shellfish Immunol. 23, 892–896.
Nikoskelainen, S., Salminen, S., Bylund, G., Ouwehand, A., 2001. Characterization of the
properties of human and dairy-derived probiotics for prevention of infectious diseases
in fish. Appl. Environ. Microbiol. 67, 2430–2435.
Oggioni, M.R., Ciabattini, A., Cuppone, A.M., Pozzi, G., 2003. Bacillus spores for vaccine
delivery. Vaccine 21 (Suppl. 2), S96–S101.
Ouwehand, A., Vankerckhoven, V., Goossens, H., Huys, G., Swings, J., Vancanneyt, M.,
Lähteenmäki, A., 2005. The safety of probiotics in foods in Europe and its legislation.
In: Goktepe, I., Juneja, V.K., Ahmedna, M. (Eds.), Probiotics in Food Safety and
Human Health. CRC Press, pp. 405–429.
Pridgeon, J., 2012. CAB Rev. 7 (048).
Pridgeon, J.W., Klesius, P.H., 2011. Development and efficacy of novobiocin and ri-
fampicin-resistant Aeromonas hydrophila as novel vaccines in channel catfish and Nile
tilapia. Vaccine 29, 7896–7904.
Raida, M.K., Larsen, J.L., Nielsen, M.E., Buchmann, K., 2003. Enhanced resistance of
rainbow trout, Oncorhynchus mykiss (Walbaum), against Yersinia ruckeri challenge
following oral administration of Bacillus subtilis and B. licheniformis (BioPlus2B). J.
Fish Dis. 26 (8), 495–498.
Rajashekhar, M., Shahanaz, E., Vinay, K., 2017. Biochemical and molecular character-
ization of Bacillus spp. isolated from insects. J. Entomol. Zool. Stud. 5 (5), 581–588.
Rajeswari, S.B.R., Shome, Y., Mazumder, A., Das, A., Kumar, H., Rahman, Bujarbaruah,
K.M., 2005. Abdominal dropsy disease in major carps of Meghalaya: isolation and
characterization of Aeromonas hydrophila. Curr. Sci. 88, 1897–1900.
Ramakrishnan, C.M., Haniffa, M.A., Manohar, M., Dhanaraj, M., Arockiaraj, A.J.,
Arunsingh, S.V., 2008. Effects of probiotics and spirulina on survival and growth of
juvenile common carp (Cyprinus carpio). Isr. J. Aquacult. 60, 128–133.
Ramesh, D., Vinothkanna, A., Rai, A.K., Venkada Subramanian, V., 2015. Isolation of
potential probiotic Bacillus spp. and assessment of their subcellular components to
induce immune responses in Labeorohita against Aeromonas hydrophila. Fish Shellfish
Immunol. 45, 268–276.
Ran, C., Carrias, A., Williams, M.A., Capps, N., Dan, B.C.T., Newton, J.C., Kloepper, J.W.,
Ooi, E.L., Browdy, C.L., Terhune, J.S., Liles, M.R., 2012. Identification of Bacillus
strains for biological control of catfish pathogens. PLoS One 7, e45793. https://doi.
org/10.1371/journal.pone.0045793.
Ray, A.K., Roy, T., Mondal, S., Ringø, E., 2010. Identification of gut-associated amylase,
cellulase, and protease-producing bacteria in three species of Indian major carps.
Aquacult. Res. 41, 1462–1469.
Ray, A.K., Ghosh, K., Ringø, E., 2012. Enzyme-producing bacteria isolated from fish gut: a
review. Aquacult. Nutr. 18, 465–492.
Reddy, J., 2015. Probiotics in aquaculture: importance, influence and future perspectives.
Int. J. Bioassays 4, 3710–3718.
Rendueles, O., Kaplan, J.B., Ghigo, J.M., 2013. Antibiofilm polysaccharides. Environ.
Microbiol. 15, 334–346.
Rengpipat, S., Rukpratanporn, S., Piyatiratitivorakul, S., Menasveta, P., 1998. Probiotics
in aquaculture: a case study of probiotics for larvae of the black tiger shrimp (Penaeus
monodon). In: Flegel, T.W. (Ed.), Advances in Shrimp Biotechnology. National Center
for Genetic Engineering and Biotechnology, Bangkok.
69
Aquaculture Reports 11 (2018) 59–69
Rengpipat, S., Rukpratanporn, S., Piyatiratitivorakul, S., Menasaveta, P., 2000. Immunity
enhancement on black tiger shrimp (Penaeus monodon) by a probiont bacterium
(Bacillus S11). Aquaculture 191, 271–288.
Resende, J., Silva, V., Fontes, C., Souza-Filho, J., Oliveira, T., Coelho, C., Cesar, D., Diniz,
C., 2012. Multidrug-resistance and toxic metals tolerance of medically important
bacteria isolated from an aquaculture system. Microbes Environ. 27 (4), 449–455.
Rinkinen, M., Jalava, K., Westermarck, E., Salminen, S., Ouwehand, A.C., 2003.
Interaction between probiotic lactic acid bacteria and canine enteric pathogens: a risk
factor for intestinal Enterococcus faecium colonization. Vet. Microbiol. 92, 111–119.
Robins-Browne, R.M., Levine, M.M., 1981. The fate of ingested Lactobacilli in the prox-
imal small intestine. Am. J. Clin. Nutr. 34, 514–519.
Ryan, K.J., Ray, C.G. (Eds.), 2014. Medical Microbiology, vol. 961. McGraw-Hill Educ, pp.
407–432.
Saikot, F.K., Zaman, R., Khalequzzaman, M., 2013. Pathogenecity test of Aeromonas
isolated from motile Aeromonas septicemia (MAS) infected Nile tilapia on some
freshwater fish. Sci. Int. 1 (9), 325–329.
Sanders, M.E., Morelli, L., Tompkins, T.A., 2003. Compr. Rev. Food Sci. Food 2 (3),
101–110.
Sariati, W.N.E., Amanu, K.S., Widayanti, R., 2015. Phenotypic and genotypic comparison
of Pseudomonas stutzeri in freshwater fish in Indonesia. J. Agric. Sci. Technol. B 5,
292–296.
Schaper, K.J., Schubert, S., Dalhoff, A., 2005. Kinetics and quantification of antibacterial
effects of beta-lactams, macrolides, and quinolones against gram-positive and gram-
negative RTI pathogens. Infection 33, 3–14.
Schillinger, U., Lucke, F.R., 1989. Antibacterial activity of Lactobacillus sake isolated from
meat. Appl. Environ. Microbiol. 55, 1901–1906.
Sebastiao, F.A., Furlan, L.R., Hashimoto, D.T., Pilarski, F., 2015. Identification of bacterial
fish pathogens in brazil by direct colony PCR and 16S rRNA gene sequencing ENC.
Adv. Microbiol. 5, 409–424.
Selim, K.M., Reda, R.M., 2015. Improvement of immunity and disease resistance in the
Nile tilapia, Oreochromis niloticus, by dietary supplementation with
Bacillusamyloliquefaciens. Fish Shellfish Immunol. 44, 496–503.
Shah, K., Mody, K., Keshri, J., Jha, B., 2010. Purification and characterization of a sol-
vent, detergent and oxidizing agent tolerant protease from Bacillus cereus isolated
from the Gulf of Khambhat. J. Mol. Catal. B: Enzym. 67, 85–91.
Sharifuzzaman, S.M., Austin, B., 2009. Influence of probiotic feeding duration on disease
resistance and immune parameters in rainbow trout. Fish Shellfish Immunol. 27,
440–445.
Sim, J.H., Jamaludin, N.S., Khoo, C.H., Cheah, Y.-K., Abdul Halim, S.N.B., Seng, H.-L.,
Tiekink, E.R.T., 2014. In vitro antibacterial and time-kill evaluation of phosphane-
gold(I) dithiocarbamates, R3Pau [S2CN(iPr)CH2CH2OH] for R = Ph, Cy and Et,
against a broad range of gram-positive and gram-negative bacteria. Gold Bull. 47,
225–236.
Smith, L.S., 1989. Digestive functions in teleost fishes. In: Halver, J.E. (Ed.), Fish
Nutrition. Academic Press, San Diego, CA, USA, pp. 331–421.
Stoodley, P., Sauer, K., Davies, D.G., Costerton, J.W., 2002. Biofilms as complex differ-
entiated communities. Ann. Rev. Microbiol. 56, 187–209.
Sugita, H., Shibuya, K., Shimooka, H., Deguchi, Y., 1996. Antibacterial abilities of in-
testinal bacteria in freshwater cultured fish. Aquaculture 145, 195–203.
Swapna, H.C., Amit, K.R., Bhaskar, N., Sachindra, N.M., 2010. Lipid classes and fatty acid
profile of selected Indian fresh water fishes. J. Food Sci. Technol. 47, 394–400.
Thankappan, B., Ramesh, D., Ramkumar, S., Natarajaseenivasan, K., Anbarasu, K., 2015.
Characterization of Bacillus spp. from the gastrointestinal tract of Labeo rohita—to-
wards to identify novel probiotics against fish pathogens. Appl. Biochem. Biotechnol.
175, 340–353.
Thapa, N., Pal, J., Tamang, J.P., 2004. World J. Microbiol. Biotechnol. 20 (6), 599–607.
Uyar, F., Porsuk, I., Kizil, G., Yilma, E., 2011. Optimal conditions for production of ex-
tracellular protease from newly isolated Bacillus cereus strain CA15. EurAsian J.
BioSci. 5, 1–9.
Verma, R.K., Jakhar, V., Gahlawat, S.K., 2006. Epizootic ulcerative syndrome in fresh
water fishes in Haryana. In: Proceedings of the 21stNational Symposium of Indian
Society of Life Science on Recent Advances in Life Sciences and Environmental
Conservation in Welfare of Human Society. K.U Kurukshetra. pp. 67–68.
Verschuere, L., Rombaut, G., Sorgeloos, P., Verstraete, W., 2000. Probiotic bacteria as
biological control agents in aquaculture. Microbiol. Mol. Biol. Rev. 64, 655–671.
Vivas, J., Carracedo, B., Riaño, J., Razquin, B.E., López-Fierro, P., Acosta, F., Naharro, G.,
Villena, A.J., 2004. App. Environ. Microbiol. 70 (5), 2702–2708.
Wang, Y.B., Li, J.R., Lin, J., 2008. Probiotics in aquaculture: challenges and outlook.
Aquaculture 281, 1–4.
Welker, T.L., Sheomaker, C.A., Arias, C.R., Kleius, P.H., 2005. Transmission and detection
ofFlavobacterium columnare in channel catfish Ictalurus punctatus. Dis. Aquat. Organ.
63, 129–138.
Zuberi, S.N., Nadeem, S.G., 2017. Detection of biofilm forming ability of bacterial isolates
from contact lenses and their accessories. J. Bacteriol. Parasitol. 8, 321.