Antagonism against fish pathogens by cellular components and verification of probiotic properties in autochthonous bacteria isolated from the gut of an Indian major carp,Catla catla(Hamilton)

Antagonism against fish pathogens by cellular

components and verification of probiotic properties in

autochthonous bacteria isolated from the gut of an

Indian major carp, Catla catla (Hamilton)

Anjan Mukherjee & Koushik Ghosh

Aquaculture Laboratory, Department of Zoology, The University of Burdwan, Golapbag, Burdwan, West Bengal

713 104, India

Correspondence: Dr K Ghosh, Aquaculture Laboratory, Department of Zoology, The University of Burdwan, Golapbag, Burdwan

713104, West Bengal, India. Emails: [email protected]; [email protected]

Abstract

Probiotic potential of the autochthonous bacteria

in catla, Catla catla has been evaluated through

determination of antagonistic activity (in vitro) of

the cellular components of gut bacteria against

seven fish pathogens. Altogether 208 strains

were isolated, inhibitory activity of the isolates

was evaluated through cross-streaking and 16

primarily selected antagonistic strains were con-

firmed using the double-layer method. Four bac-

teria that showed antagonism against ≥4pathogens were selected as putative probiotics.

The intracellular, extracellular, whole-cell and

heat-killed cell components exhibited bactericidal

activity against the pathogens. In addition, the

selected strains were capable of producing differ-

ent extracellular enzymes, competent to grow in

intestinal mucus and could tolerate diluted bile

juice. Analysis of 16SrRNA partial gene sequence

revealed that both the strains CC1FG2 and

CC1FG4 were Bacillus methylotrophicus (KF559344

and KF559345), while the isolates CC1HG5 and

CC2HG7 were Bacillus subtilis subsp. spizizenii

(KF559346) and Enterobacter hormaechei

(KF559347) respectively. Bio-safety evaluation

through intra-peritoneal injection of the isolates

did not induce any pathological signs or mortali-

ties in C. catla. The study confirmed probiotic prop-

erties of autochthonous gut bacteria in C. catla

and demonstrated potential for using them as bio-

control agents. However, in vivo studies are essen-

tial to explore their efficacy in the commercial

aquaculture.

Keywords: antagonism, Bacillus, Catla, cellular

components, Enterobacter, gut bacteria, probiotic

characterization

Introduction

Bacteria are the most common among the patho-

gens in cultured fish that cause mass mortality in

freshwater aquaculture (Swain, Behura, Dash &

Nayak 2007; Giri, Sukumaran, Sen, Vinumonia,

Nazeema-Banu & Jena 2011). Antibiotics are com-

monly used to treat the bacterial diseases. Besides

causing environmental problems (Martinez 2009),

the excessive and improper use of antibiotics in

aquaculture has led to the development of drug-

resistant bacteria that are becoming increasingly

difficult to control and eliminate (Bruun, Schmidt,

Madsen & Dalsgaard 2000; Nomoto 2005). In

addition, the use of antibiotics is also under criti-

cism due to the accumulation of residues in fish

tissues (Chevassus & Dorson 1990). Therefore,

development of alternative ways of combating dis-

eases avoiding the use of antibiotics has been

necessitated worldwide (Nogami & Maeda 1992;

Sugita, Hirose, Matsuo & Deguchi 1998; Gate-

soupe 1999; Bala-Reddy 2001).

The production of antimicrobial substances by

some bacteria seemed to play an important role

in antagonizing other bacteria in aquatic ecosys-

tems (Dopazo, Lemos, Lodeiros, Bolinches, Barja &

Toranzo 1988; Sugahara, Kimura, Hayashi & Nak-

ajima 1988). Therefore, the use of non-pathogenic

bacteria as probiotic bio-control agents has been

© 2014 John Wiley & Sons Ltd 1

Aquaculture Research, 2014, 1–13 doi:10.1111/are.12676

proposed by several workers. Antimicrobial sub-

stances produced by bacilli isolated from intestines

of Japanese costal fish (Sugita et al. 1998) and an

Indian Major Carp, Labeo rohita (Giri et al. 2011)

have been documented as bio-control agents.

Antagonistic activities of Pseudomonas species

against Aeromonas (Das, Samal, Samantaray,

Sethi, Pattnaik & Mishra 2006; Giri et al. 2011)

and Vibrio sp. (Vijayan, Bright Singh, Jayaprak-

ash, Alavandi, Pai, Preetha, Rajan & Santiago

2006) have been reported. Consequently, a gen-

eral consensus developed that probiotic treatment

might lead to better protection of fish against

multiple diseases.

Although, the selection of probiotics for aqua-

culture might be based on several criteria, the abil-

ity to colonize the host gut is often believed to be

one of the main selection criteria for prospective

probiotics (Merrifield, Harper, Dimitroglou, Ringø

& Davies 2010; Nayak 2010). Perhaps, growth on

mucus and tolerance to the bile salts are the deci-

sive factors for colonization of any gut microor-

ganism. Therefore, emphasis has been given on

the autochthonous microorganisms to search for

beneficial bacteria (Fjellheim, Klinkenberg,

Skjermo, Aasen & Vadstein 2010), as the native

flora are supposed to be well adapted to the

intended ecological niche (O’Sullivan 2001). Previ-

ous studies have isolated diverse bacteria species

from the GI tract of Indian major carps, exotic

carps and other cultivable teleosts, and probable

beneficial roles of the gut microbiota pertaining to

nutrition of the host fish have been emphasized

(Ghosh, Sen & Ray 2002a; Kar, Roy, Sen & Ghosh

2008; Ray, Roy, Mondal & Ringø 2010; Ghosh,

Roy, Kar & Ringø 2010; Khan & Ghosh 2012; for

review see Ray, Ghosh & Ringø 2012). Further-

more, attempts have also been made to use benefi-

cial gut bacilli originally isolated from rohu as the

probiotics for the fish (Ghosh, Sen & Ray 2002b,

2003). However, antimicrobial potential of the

beneficial gut bacteria together with challenge

studies have been rarely carried out in tropical

freshwater fish to inhibit the potential fish patho-

gens. Therefore, the presently reported study con-

sidered in vitro antagonistic activity of the cellular

components of the potential probiotic bacteria iso-

lated from the gut of an Indian Major Carp, catla

(Catla catla) Furthermore, characterization of the

probiotic properties in the antagonistic bacteria

has been complemented by their ability to produce

extracellular enzymes, growth in fish mucus, resis-

tance to bile juice, and safety evaluation for the

target fish.

Materials and methods

Collection and processing of sample

Specimens of the Indian major carp, catla, Catla cat-

la (Hamilton) were collected from three composite

carp culture ponds located at and around Burdwan

(23°140N, 87°390E), West Bengal, India. The ranges

of water quality parameters during the collection

period were: dissolved oxygen 6.5–7.8 mg L�1, pH

6.8–7.2 and temperature 26.2–27.8°C. Health

status of the fish was checked following Smith,

Donahue, Lipkin, Blazer, Schmitt and Goede (2002)

and 3 living specimens of grow-out C. catla with no

external anomalies, lesions or disease symptoms

were collected from each collection pond, and

thus altogether nine fish (average live weight

245 � 10.84 g) were collected, brought to the lab-

oratory with oxygen packing and distributed sepa-

rately over 3 aquaria of 75 L on the basis of their

source. The fish were kept in starvation for 48 h

prior to sacrifice in order to clear their GI tracts.

After starvation, the fish were anaesthetized

by applying 0.03% of tricaine methanesulfonate

(MS-222; Sigma-Aldrich Corp., St. Louis, MO, USA),

the ventral surfaces were sterilized using 70% etha-

nol and dissected aseptically to remove the intestine

(Ghosh et al. 2010). Gut samples were divided into

PI (proximal intestine) and DI (distal intestine), and

processed according to Mandal and Ghosh (2013)

for isolation of autochthonous microorganisms. Gut

segments from three specimens of C. catla were

pooled together region-wise for each replicate, and

thus there were three replicates for the study. To

keep away from erroneous conclusions due to indi-

vidual disparity in gut microbiota, pooled samples

of 3 fish were used for each replicate as described

elsewhere (Ringø, Strøm & Tabachek 1995).

Isolation of bacterial strains

Homogenates of the intestinal segments were seri-

ally diluted (1:10) in 0.9% normal saline (Beve-

ridge, Sikdar, Frerichs & Millar 1991) and

aseptically plated onto Soyabean Casein Digest

Medium (Tryptone Soya Agar, TSA; HiMedia

Laboratories Pvt Ltd., Mumbai, India) at 30°C for

48 h to isolate the autochthonous culturable het-

erotrophic aerobic/facultative anaerobic bacteria

© 2014 John Wiley & Sons Ltd, Aquaculture Research, 1–132

Pathogen inhibitory gut bacteria in Catla A Mukherjee & K Ghosh Aquaculture Research, 2014, 1–13

population. The well-separated colonies were ran-

domly collected and streaked separately on TSA

plates to obtain pure cultures, transferred to TSA

slants and maintained in a refrigerator (4°C) for

further study.

Pathogen collection and culture conditions

Four fish pathogenic strains Aeromonas salmonicida

MTCC-1945 (AS), Pseudomonas fluorescens MTCC-

103 (PF), Pseudomonas putida MTCC-1072 (PP)

and Bacillus mycoides MTCC-7538 (BM) were

obtained from the Microbial Type Culture Collec-

tion, Chandigarh, India. In addition to A. salmoni-

cida and P. fluorescens, both Pseudomonas putida

(Altinok, Kayis & Capkin 2006) and Bacillus my-

coides (Goodwin, Spencer Roy, Grizzle & Goldsby

1994) are described as opportunistic fish patho-

gens. Three other strains, Aeromonas hydrophila

(AH), Aeromonas veronii (AV) and Pseudomonas

sp. (P) were isolated from diseased fish. Pathoge-

nicity of the isolated strains was checked experi-

mentally by intraperitoneal injection to C. catla

and by observing the onset of disease in the fish.

The pathogenic strains were maintained in the

laboratory on TSA (HiMedia) slants at 4°C. Stockcultures in tryptone soya broth (TSB) were stored

at �70°C in 0.85% NaCl with 20% glycerol to

provide stable inoculums throughout the study

(Sugita et al. 1998).

Assay for pathogen inhibitory activity

Inhibitory activity of the isolated strains against

the said seven fish pathogens was primarily

noticed through ‘cross-streaking’ (Madigan,

Martiko & Parker 1997). Later, antagonistic activ-

ity of the primarily selected strains was confirmed

using the ‘double-layer’ method (Dopazo et al.

1988) with minor modification. Briefly, putative

antagonistic strains were grown on TSA plates at

30°C for 48 h, the cells were killed with chloro-

form vapour (15 min), overlaid with the cultures

containing the pathogenic strains and further

incubated for 48 h at 30°C. There were three rep-

licates for each experimental set. A clear zone of

inhibition (halo) around growth of the selected gut

bacteria indicated antibacterial activity and the

halo zone (diameter in mm) around the colony

was presented as scores as follows; 0 (0–5 mm), 1

(low, 6–10 mm), 2 (moderate, 11–20 mm), 3

(high, 21–25 mm) and 4 (very high, ≥26 mm).

Preparation of different cellular components

Different cellular components, e.g. the whole-cell prod-

uct (WCP), heat-killed whole-cell product (HKWCP),

intracellular product (ICP), and extracellular product

(ECP) were prepared from the four selected strains

following Das et al. (2006). Pure cultures were

maintained separately under sterile conditions in

400 mL TSB (pH 7.0) (HiMedia) at 30°C for 24 h,

sub-divided into 4 equal volumes of 100 mL and

used for preparation of the WCP, HKWCP, ICP

and ECP. The bacterial strains grown in TSB for

24 h were centrifuged at 10 000 g for 10 min at

4°C. The pellets obtained were washed twice and

re-suspended in PBS (pH 7.2) for use as WCP.

After centrifugation, heat-killed pellets (at 60°C for

1 h in water bath) were used as HKWCP. ICP

were prepared by sonication (50 Hz, 5 min) of the

re-suspended pellets in PBS (pH 7.2, 2% of the ini-

tial volume) and filtration through a syringe with

a 0.45 lL filter. Supernatants obtained after the

centrifugation of the TSB cultures were filtered

through a 0.22 lm filter (HiMedia) and concen-

trated with a freeze dryer to use as ECP. ECP from

the selected bacteria ranged between pH 7.0–7.2.All cellular components were stored at �20°Cuntil use. The protein content of the cellular com-

ponents was estimated by Lowry, Rosenbrough,

Fair and Randall (1951). The minimum amount

of protein content (100 lL) of the lowest protein

concentration (ECP of CC1FG4) was taken as stan-

dard and accordingly the other protein contents

were adjusted. Cellular components were charged

into the agar wells on the TSA media plates to

study antagonism against the pathogenic strains.

Study of antagonistic activity using cellular

components

Fish pathogenic strains (24 h old cultures) were

inoculated (108 cells 100 lL�1) by pour plating

and separately grown on TSA media (Tryptone

Soya broth supplemented with 1% agar; HiMedia)

plates. Agar wells (6 mm dia.) were prepared,

aliquots of different cellular components (100 lL)were poured in the respective wells and incu-

bated for 24 h at 30°C. Appearance of zones of

inhibition (halo, diameter in mm) around the

wells were recorded and presented accordingly.

For each cellular component, averages for 3 wells

in each plate were calculated and triplicate plates

were maintained along with the sterile PBS (pH

© 2014 John Wiley & Sons Ltd, Aquaculture Research, 1–13 3

Aquaculture Research, 2014, 1–13 Pathogen inhibitory gut bacteria in Catla A Mukherjee & K Ghosh

7.2) control. The zones of inhibition produced by

the antibiotics, viz. chloramphenicol (30 lg) and

gentamicin (15 lg), against the fish pathogens

were treated as positive controls (Giri et al.

2011).

Determination of extracellular enzyme production

Quantitative assay of the extracellular enzyme

activity in the 4 bacterial isolates selected through

antagonistic activity were performed. Selective

broth media were used as production media for

the enzymes (viz., amylase, protease, cellulase,

lipase, xylanase and phytase) and the contents

were centrifuged at 10 000 g for 10 min, at 4°C.The cell-free supernatant was used as the source

of enzyme (Bairagi, Ghosh, Sen & Ray 2002).

Amylase and cellulase activities were determined

following the methods using dinitro-salicylic-acid

described by Bernfeld (1955) and Denison and

Koehn (1977) respectively. Protease activity was

determined using the casein digestion method of

Walter (1984). Lipase activity was measured fol-

lowing the method described by Bier (1955).

Quantitative assay of xylanase and phytase activi-

ties were measured after Bailey, Biely and Pouta-

nen (1992) and Yanke, Selinger and Cheng

(1999), respectively, using birchwood xylan and

Na-phytate as substrates. Protein content of the

enzyme source was measured after Lowry et al.

(1951) and quantitative enzyme activities were

expressed as units (U).

Growth on fish mucus

Mucus collection

Mucus from fish skin was collected after Ross,

Firth, Wang, Burka and Johnson (2000). Briefly,

three fish of C. catla were anaesthetized with

MS-222 and positioned in individual plastic bags

containing 5 mL of 100 mM ammonium bicar-

bonate (NH4HCO3), pH 7.8 for 10 min. At the

time of removal of fish, an additional 5 mL of

buffer were added and the bags were placed on

ice. For intestinal mucus, intestine was dissected

out, flashed twice with PBS, mucus was scrubbed

and homogenized with 100 mM NH4HCO3. To

remove copepods and cellular or other foreign

materials, mucus samples were transferred into

sterile 15 mL tubes, centrifuged at 12 000 g for

15 min at 4°C. Protein concentration of the

mucus preparations were determined after Lowry

et al. (1951) and adjusted to a concentration of

1 mg mL�1 for use as growth media. Samples

were then filter-sterilized through 0.8 and

0.22 lm pore size filter paper (HiMedia) and

stored at �80°C until use.

Standard plate count (viable counts)

Filter-sterilized mucus samples were inoculated

(107 CFU mL�1) with the four antagonistic strains

separately and grown at 30°C for 24 h. Serial

dilutions (10�3, 10�4 and 10�5) of the bacterial

cultures in mucus were made, inoculated (0.1 mL)

on to TSA plates, incubated for 24 h at 30°C and

colony-forming units (CFU mL�1) were counted.

Plates inoculated with sterilized mucus were

served as controls.

Bile tolerance

Bile juice (pH 5.7) was collected from dissected gall

bladders in aseptic condition, filter sterilized

through 0.8 and 0.22 lm pore size filter papers (Hi-

Media) and stored at �20°C until use. Bacteria

grown in TSB for 24 h were centrifuged at

10 000 g for 10 min at 4°C and bacterial suspen-

sions were prepared in PBS. The bacterial suspen-

sion was inoculated (107 CFU mL�1) in sterile PBS

(control) or in sterile PBS supplemented with

2–20% (v/v) fish bile juice, as described elsewhere

(Nikoskelainen, Salminen, Bylund & Ouwehand

2001; Balcazar, Vendrell, De Blas, Ruiz-Zarzuela,

Muzquiz & Girones 2008). Following incubation of

the bacteria samples for 1.5 h at 30 °C, samples

were serially diluted in sterile PBS and viable bacte-

ria counts were determined on TSA media plates.

Safety evaluation of potential probiotic bacteria

Bio-safety evaluation of the four putative probionts

was carried out through in vivo studies conducted

in 75 L glass aquaria. Healthy fingerlings of C. catla

(15 � 1.2 g) were acclimatized in the laboratory

condition for 2 weeks. Seventy five fish were divided

into five groups (four experimental and one con-

trol), each with three replicates. The candidate pro-

biotics were grown in TSB at 30°C for 24 h,

centrifuged (2800 g for 15 min, at 4°C) and cell

pellets were suspended in sterile 0.9% saline. Experi-

mental fish were injected intraperitoneally (IP) with

1.0 mL of suspension containing 109 cells mL�1

(determined using Petroff-Hausser counting

chamber) of a candidate probiotic bacteria. The fish



in control group were injected with sterile 0.9% sal-

ine (Mesalhy, Abd-El-Rahman, John & Mohamed

2008). Fish were fed ad libitum with a diet contain-

ing approximately 40% crude protein having fish

meal as the chief protein source. All groups were

kept under observation for 21 days and health sta-

tus was recorded every day.

Identification of isolates by 16S rRNA partial gene

sequence analysis

Four most potent antagonistic bacteria were exam-

ined and identified through 16S rRNA partial gene

sequence analysis after isolation and PCR amplifica-

tion following the methods described in Das, Man-

dal, Khan, Manna and Ghosh (2014). The gene

encoding 16S rRNA was amplified from the isolates

by polymerase chain reaction (PCR) using universal

primers 27f (50-AGAGTTTGATCCTGGCTCAG-30)and 1492r (50-GGTTACCTTGTTACGACTT-30). ThePCR reactions were performed using PCR mix con-

taining 200 lM of deoxynucleotides (dNTPs),

0.2 lM of each primer, 2.5 mM MgCl2, 19 PCR

buffer and 0.2 U of Taq DNA polymerase (Invitro-

gen Corp., Carlsbad, CA, USA). The template DNA

was obtained using extracting genomic DNA using

Gen EluteTM Bacterial genomic DNA Kit (Sigma-

Aldrich) from a fresh colony grown on nutrient

agar slant. The cycle used for PCR reaction was:

denaturation at 95°C for 3 min followed by 35

cycles of 95°C for 1 min, annealing at 55°C for

1 min, extension at 72°C for 2 min and a final

extension at 72°C for 3 min (Lane 1991). PCR

products were sent to the commercial house for

Sanger sequencing using automated DNA sequen-

cer (Applied Biosystems, Inc., Foster City, CA, USA).

Sequenced data were edited using BioEdit Sequence

Alignment Editor (Version 7.2.0), aligned and anal-

ysed for finding the closest homologue using

National Centre for Biotechnology Information

(NCBI) GenBank and Ribosomal Database Project

(RDP) databases. Sequences were deposited to the

NCBI GenBank to obtain accession numbers and a

phylogenetic tree was constructed incorporating

16S rRNA partial gene sequences of the closest type

strains using MEGA 5.1 Beta4 software following

the Minimum Evolution Method.

Statistical analysis

The differences in the inhibition profile by the cellu-

lar components from each of the 4 selected probi-

onts (CC1FG2, CC1FG4, CC1HG5 and CC2HG7)

were assessed separately against the fish pathogens

(AH, AV, AS, P, PF, PP) through one way ANOVA,

followed by Tukey’s test. Data pertaining to extra-

cellular enzyme production were also subjected to

one way ANOVA and Tukey’s test. Non-parametric

Friedman’s test was performed to compare growth

of the tested strains in skin and intestinal mucus.

The statistical analyses were carried out following

Zar (1999) using SPSS version 10 (Kinnear & Gray

2000) software.

Results

Isolation of gut bacteria and determination of

pathogen inhibitory activity

Analysis of microbial population revealed that bac-

teria antagonistic to the tested fish pathogens exist

in the proximal (PI) and distal (DI) segments of the

GI tracts of C. catla. Altogether 208 strains were

isolated (94 from PI and 114 from DI regions) from

the GI tracts, out of which 16 strains (07 from PI

and 09 from DI regions, 7.69% of the total isolates)

were noticed with inhibitory activity against 2 or

more selected fish pathogens by ‘cross-streaking’.

Antagonistic activity of these primarily selected 16

strains against the tested 7 pathogens was further

evaluated through the double-layer method and

presented as scores (Table 1), cumulative maxi-

mum and minimum scores being 19 and 6 respec-

tively. Based on the total scores, 4 bacterial isolates

(CC1FG2, CC1FG4, CC1HG5 and CC2HG7) that

showed inhibitory activity against ≥4 studied fish

pathogens were selected as putative probiotics and

different cellular components were analysed for

antagonism against the pathogenic strains. These

strains were also assessed for verification of the

other probiotic properties.

Protein estimation of different cellular components

of selected bacteria

Analysis of the protein contents in various cellular

components of CC1FG2, CC1FG4, CC1HG5 and

CC2HG7 revealed the highest concentration in the

ICP of CC2HG7 (2.86 � 0.012 mg mL�1), being

the lowest in the ECP of CC1FG4

(2.22 � 0.009 mg mL�1). The minimum quantity

of protein (in 100 lL) obtained in the ECP of

CC1FG4 was 222 lg. Protein contents in the other

cellular components were adjusted to 222 lg



corresponded to 100 lL and applied against the

pathogenic strains on TSA plates.

Antagonistic activity of the cellular components

The cellular components, WCP, HKWCP, ICP and

ECP from each of the 4 selected putative probiotics

showed antagonism against different pathogenic

bacteria (Table 2). Variation in the antagonistic

activity was noticed among the different cellular

components and also among the selected bacteria.

None of the selected bacteria produced antagonism

against BM, and thereby not included in the com-

parative data shown in this section.

The cellular components from the four selected

bacteria showed significant (P < 0.05) bactericidal

activity against the AH, compared with the antibi-

otics used as positive controls. Among all, WCP of

CC2HG7 produced maximum antagonistic activity

against AH (44 � 1.27 mm), whereas, minimum

inhibition zone was recorded with the ECP of

CC1FG4 against AH (31.5 � 1.00 mm). The cel-

lular components of CC1FG2, CC1FG4 and

CC2HG7 displayed antagonism against AV. In

comparison to the positive controls, significant dif-

ferences (P ≤ 0.05) in the antagonistic activities

were observed only with the cellular components

of CC1FG2. Positive controls were either more

effective (Chloramphenicol) or did not differ signifi-

cantly (Gentamicin) from the cellular components

of CC1FG4 and CC2HG7. The cellular components

of the putative probiotics produced reasonable bac-

tericidal activity against AS, being the highest

inhibition zone recorded with the WCP of CC1FG2

(42.8 � 0.92 mm). Bacterial cellular components

were either less effective (CC1HG5) than the posi-

tive controls or did not show antagonism at all

against P. Compared with the positive controls,

the cellular components exhibited significant

(P ≤ 0.05) bactericidal activity against PF, except

from CC1HG5. WCP of CC2HG7 produced the

greatest inhibition zone against PF

(43 � 0.68 mm). Among the putative probionts

studied, cellular components of CC1HG5 and

CC2HG7 elicited antagonism against PP that dif-

fered significantly from the positive controls. The

maximum inhibition zone (46.8 � 1.04 mm)

against PP was produced by the HKWCP of

CC2HG7, although it did not differ significantly

from the ICP of the same.

Determination of extracellular enzyme production

Capacity of extracellular enzyme production differed

among the selected gut bacteria antagonistic to dif-

ferent fish pathogens (Table 3). The strain CC1HG5

exhibited maximum protease (71.58 � 2.21 U)

and xylanase (20.76 � 0.98 U) activities. While

the highest amylolytic (260.10 � 5.67 U) and lipo-

lytic (4.67 � 0.36 U) activities were noticed with

the isolates CC1FG2 and CC2HG7 respectively.

Maximum cellulase (72.28 � 2.24 U) and phytase

(200.40 � 5.84 U) activities were recorded with

the strains CC1FG4 and CC2HG7 respectively. In

addition, the strain CC1FG4 showed minimum pro-

tease (52.56 � 2.09 U) and xylanase (7.24 �0.31 U) activities. The lowest cellulase (71.23 �2.06 U) and phytase (75.82 � 2.12 U) activities

were noticed with the strain CC1HG5. The isolates

CC1FG2 and CC2HG7 revealed the lowest lipolytic

(4.18 � 0.16 U) and amylolytic (233.2 � 4.92 U)

activities respectively.

Growth on fish mucus

All of the four selected gut isolates grew well in

fish mucus collected from both, skin and intestine

of C. catla (Table 4). Irrespective of the strains

Table 1 Determination of antagonism (double agar layer

method) by the primarily selected gut bacteria against

tested fish pathogens (described in the text). Zones of

inhibition (halo diameter) were presented as scores*

Strains AH AV AS P PF PP BM

Total

score

CC1FG2 4 4 4 – 4 – – 16

CC1FG4 4 3 4 – 4 15

CC1HG5 4 – 4 1 3 4 – 16

CC1HG6 1 2 4 1 2 – – 10

CC1HG7 – 4 – 4 2 3 – 13

CC2FG1 3 – 4 2 – – – 9

CC2FG2 – – – 3 – 3 – 6

CC2FG4 – – 4 – – – 3 7

CC2FG16 – 3 4 – 3 – – 10

CC2HG6 3 – 2 4 – – – 9

CC2HG7 4 3 4 – 4 4 – 19

CC3FG2 2 – – 2 – – 3 7

CC3HG8 – – – 4 3 – – 7

CC3HG11 – – 4 3 – 2 – 9

CC3HG12 3 2 – – 3 2 – 10

CC3HG18 – – 4 2 – 2 – 11

*1, low (6–10 mm); 2, moderate (11–20 mm); 3, high (21–

25 mm); 4, very high (≥26 mm). Data represents mean value

of three observations.

AH, A. hydrophila; AV, A. veronii; AS, A. salmonicida; P, Pseudo-

monas; PF, P. fluorescens, PP, P. putida; BM, B. mycoides.



studied, the growth in the mucus from two differ-

ent sources varied significantly as observed

through the non-parametric Friedman’s test

(Q = 12; d.f. = 1; P < 0.001). Thus, Log viable cell

counts (CFU mL�1) of all the studied probionts

demonstrated that the strains were more capable

to grow in intestinal mucus than the skin mucus.

Bile tolerance

All of the four candidate probiotic bacteria showed

tolerance against diluted bile juice. The selected

bacterial strains survived after 1.5 h exposure to

different concentrations of bile juice ranging pH

values 5.5–7 (Table 5). Moreover, marked changes

were not detected in the growth of the selected

bacteria even after exposure to the diluted bile

juice (2–20%) for 24 h (data not presented).

Safety evaluation of putative probiotic bacteria

After 21 days of small-scale in vivo experiment, it

was revealed that along with the control set intra-

peritoneal injection of the candidate probiotics did

not induce any pathological signs/disease symp-

toms or mortalities in all of the four treatment

groups.

Genotypic identification of the selected isolates

Based on the nucleotide homology and phyloge-

netic analysis of the 16S rRNA partial gene

sequence by nucleotide blast in the NCBI GenBank

and RDP, the strains CC1FG2 and CC1FG4 were

identified as Bacillus methylotrophicus (accession

nos. KF559344 and KF559345 respectively),

which were closest to the type strain Bacillus

methylotrophicus (EU194897.1). The isolate

CC1HG5 was identified as Bacillus subtilis subsp.

spizizenii (accession no. KF559346) that showed

maximum similarity with the type strain Bacillus

subtilis subsp. spizizenii (AF074970.1). Another

strain CC2HG7 was similar to Enterobacter hor-

maechei (accession no. KF559347) being closest

homologue to the type strain Enterobacter hormaec-

hei (AJ508302.1). Homology levels of the

Table 2 Profiles of inhibition by the cellular components of the selected gut isolate against fish pathogens

Antibacterial components

Zones of inhibition (mm) against fish pathogens

AH AV AS P PF PP

Cellular components of CC1FG2

WCP 38.2 � 0.94ef 40 � 1.35ef 42.8 � 0.92f – 38 � 0.79f –

HKWCP 36.5 � 0.35e 38.6 � 0.81e 40 � 1.13def – 39.5 � 0.85fg –

ICP 37.3 � 0.56e 42.2 � 1.21f 42.6 � 0.90f – 40.6 � 1.01g –

ECP 34 � 0.52d 36 � 0.99d 38.4 � 0.76d – 37 � 1.11ef –

Cellular components of CC1FG4

WCP 34.6 � 0.95de 25.5 � 0.72b 38.2 � 1.3d – 39.6 � 1.2fg –

HKWCP 32 � 0.96c 24.2 � 0.72ab 38 � 1.18d – 36.4 � 1.07ef –

ICP 34 � 0.98d 25 � 0.59b 39.8 � 1.06de – 40 � 0.93g –

ECP 31.5 � 1.00c 22.8 � 0.69a 35 � 1.03c – 35.2 � 1.06e –

Cellular components of CC1HG5

WCP 42.3 � 1.21g – 40.3 � 0.66e 10 � 0.85b 26 � 0.90c 38.5 � 1.44d

HKWCP 39 � 0.87f – 34.5 � 0.52c 8.5 � 0.29a 24.5 � 0.87b 37 � 0.97cd

ICP 42.5 � 0.47g – 42 � 0.92f 10 � 0.25b 26 � 0.40c 39 � 0.52d

ECP 36.8 � 1.10e – 40 � 1.09def 8 � 0.03a 22.2 � 0.46a 35.5 � 1.10c

Cellular components of CC2HG7

WCP 44 � 1.27g 26 � 0.64b 42.5 � 0.64f – 43 � 0.68h 44.5 � 0.75f

HKWCP 36.3 � 1.07e 25.5 � 0.66b 41 � 0.98ef – 38.2 � 1.04f 46.8 � 1.04g

ICP 43.5 � 1.04g 26 � 0.81b 40.5 � 0.72ef – 42 � 1.04gh 45 � 0.98fg

ECP 39.2 � 1.01f 23.8 � 0.69ab 38.3 � 0.98d – 40.5 � 0.47g 41.5 � 0.75e

Positive controls (Antibiotics)

Gentamicin 23.5 � 0.40a 26 � 0.61b 24 � 0.32a 26.2 � 0.43c 26 � 0.5c 26 � 0.61a

Chloramphenicol 26 � 0.32b 29.5 � 0.57c 26.5 � 0.40b 28.6 � 0.52d 28.5 � 0.94d 29.5 � 0.75b

Data are means � SE (n = 3). Values with the same superscripts in the same vertical column are not significantly different

(P < 0.05).

–, no antagonistic activity; AH, A. hydrophila; AV, A. veronii; AS, A. salmonicida; P, Pseudomonas; PF, P. fluorescens, PP, P. putida.



identified strains were >99% with their related

type strains. Phylogenetic relation of the four iden-

tified bacterial isolates with other closely related

strains retrieved from RDP type strains are pre-

sented in the dendogram (Fig. 1).

Discussion

Reports on pathogen inhibitory gut bacteria in the

Indian Major Carps are scarce (Ghosh, Sinha &

Sahu 2007; Giri et al. 2011; Nayak & Mukherjee

2011). In the present study, altogether 208

autochthonous bacterial strains obtained from the

GI tract of an Indian Major Carp, C. catla, were

screened for indigenous candidate probiotics that

led us to the detect 16 antagonistic bacterial

strains (7.69%). Considering antagonism towards

pathogens and verification of other probiotic prop-

erties, 4 bacterial isolates (CC1FG2, CC1FG4,

CC1HG5, and CC2HG7) were characterized as

putative probiotics. The isolate CC1HG5 was iden-

tified as Bacillus subtilis subsp. spizizenii, whereas,

both the strains CC1FG2 and CC1FG4 were similar

to Bacillus methylotrophicus. Gut microbiota in the

freshwater teleosts were fairly dominated by Bacil-

lus spp. (e.g. Ghosh et al. 2010; Kar et al. 2008;

Ray et al. 2010; Mondal, Roy & Ray 2010; Ghosh

et al. 2010), which were in agreement with the

present study as 3 out of the 4 identified gut bac-

teria from C. catla were Bacilli. Previously, probiot-

ic B. subtilis BT23 and Bacillus spp. were

documented to control the growth of pathogenic

Vibrio harveyi, both in vitro and in vivo (Vaseeha-

ran & Ramasamy 2003; Janarthanam, George,

John & Jeyaseelan 2012). In another study,

Table 3 Extracellular enzyme production by the selected bacteria

Strains Amylase (U)* Protease (U)† Lipase (U)‡ Cellulase (U)§ Phytase (U)¶ Xylanase (U)**

CC1FG2 260.10 � 5.67b 53.53 � 2.12a 4.18 � 0.16a 71.64 � 2.07a 199.62 � 4.92b 7.59 � 0.26a

CC1FG4 255.44 � 5.63a 52.56 � 2.09a 4.38 � 0.23a 72.28 � 2.24a 198.33 � 4.67b 7.24 � 0.31a

CC1HG5 239.45 � 5.31a 71.58 � 2.21b 4.32 � 0.20a 71.23 � 2.06a 75.82 � 2.12a 20.76 � 0.98b

CC2HG7 233.20 � 4.92a 70.64 � 2.19b 4.67 � 0.36b 72.08 � 2.19a 200.40 � 5.84b 20.33 � 1.06b

Data are means � SE (n = 3). Values with the same superscripts in the same vertical column are not significantly different

(P < 0.05).

*Amylase (lg maltose liberated mL�1 of enzyme extract min�1).

†Protease (lg tyrosine liberated mL�1 of enzyme extract min�1).

‡Lipase (lmole free fatty acid liberated mL�1 of enzyme extract min�1).

§Cellulase (mg glucose liberated mL�1 of enzyme extract min�1).

¶Phytase (lg inorganic phosphate liberated mL�1 of enzyme extract min�1).

**Xylanase (mg D-xylose liberated mL�1 of enzyme extract min�1).

Table 4 Log values of viable count (CFU mL�1) of the

selected gut bacteria* grown in skin and intestinal mucus

of Catla catla. Viable count was done on TSA plates inoc-

ulated with respective bacteria cultures of 24 h duration

in fish mucus

Strains

Log CFU mL�1

Skin

mucus

Intestinal

mucus

CC1FG2 7.50 � 0.01 7.65 � 0.01

CC1FG4 7.48 � 0.01 7.56 � 0.04

CC1HG5 7.42 � 0.01 7.61 � 0.01

CC2HG7 7.58 � 0.01 7.79 � 0.01

*Initial count: 6 Log CFU mL�1 mucus.

Data are mean � SE (n = 3). No growth detected on plates

inoculated with sterilized mucus.

Table 5 Tolerance of the selected gut bacteria at differ-

ent concentrations of fish bile juice for 1.5 h at 30°C.

Viable count was determined on TSA plates inoculated

with bile exposed bacterial suspension

Bile

(%)

Log CFU mL�1

CC1FG2 CC1FG4 CC1HG5 CC2HG7

0 7.08 � 0.01 7.05 � 0.01 7.01 � 0.02 7.10 � 0.02

2 7.04 � 0.01 7.02 � 0.01 6.99 � 0.01 7.08 � 0.01

4 7.03 � 0.01 7.01 � 0.01 6.98 � 0.01 7.06 � 0.01

6 7.02 � 0.01 7.00 � 0.01 6.96 � 0.01 7.06 � 0.01

8 7.01 � 0.01 6.99 � 0.01 6.95 � 0.01 7.04 � 0.01

10 6.99 � 0.01 6.97 � 0.01 6.94 � 0.01 7.04 � 0.01

12 6.98 � 0.01 6.96 � 0.01 6.93 � 0.01 7.02 � 0.01

14 6.96 � 0.01 6.94 � 0.01 6.92 � 0.01 7.00 � 0.01

16 6.94 � 0.01 6.93 � 0.01 6.91 � 0.01 6.99 � 0.01

18 6.93 � 0.01 6.91 � 0.01 6.89 � 0.01 6.97 � 0.01

20 6.90 � 0.01 6.88 � 0.01 6.87 � 0.01 6.94 � 0.01

Data are mean � SE (n = 3).



B. subtilis SG4 isolated from C. mrigala was

recorded with positive antibacterial activity against

pathogenic P. fluorescens, A. hydrophila and

E. tarda (Ghosh et al. 2007). However, likely probi-

otic activity of B. methylotrophicus has been docu-

mented much later. Chao, Carrias, Williams,

Capps, Dan, Newton, Kloepper, Ooi, Browdy, Terh-

une and Liles (2012) reported B. methylotrophicus

strains from soil or channel catfish intestine and

screened for antagonism against fish pathogens

Edwardsiella ictaluri and Aeromonas hydrophila,

causing enteric septicaemia of catfish and motile

aeromonad septicaemia respectively. In the present

study, another isolate CC2HG7 was identified as

Enterobacter hormaechei. To the authors’ knowl-

edge, previously only one study evidenced screen-

ing of probiotic E. hormaechei BAC 1010 capable

of inhibiting growth of fish, shrimp and human

pathogens in vitro (Ghosh, Ringø, Deborah,

Mujeeb-Rahiman & Hatha 2011).

In the present study, the selected isolates from

the gut of C. catla were antagonistic to 4 or 5 of

the studied fish pathogens that included A. hydro-

phila, A. salmonicida and P. fluorescens. The anti-

bacterial effect of bacteria has been generally

recognized due to either individual or combined

production of antibiotics, bacteriocins, siderophores

(Gram & Melchiorsen 1996), lysozymes and prote-

ases and modification of pH by organic acid

production (Sugita et al. 1998). In addition, com-

petitive exclusion by the probiotic organism lead-

ing to inhibition of growth rate of the pathogenic

bacteria has also been proposed by several authors

(Lalloo, Moonsamy, Ramchuran, G€orgens & Gard-

iner 2010; Rico-Mora, Voltolina & Villaescusa-Ce-

laya 1998). The methods used in the presently

reported study to assay the inhibitory effect of the

putative probiotics (cross-streaking and double

layer) detect the influence of diffusing antimicro-

bial substances on the growth of pathogenic bacte-

ria. Thus, besides competitive exclusion, our

results might indicate secretion of the antibacterial

substances by B. methylotrophicus, B. subtilis

subsp. spizizenii and E. hormaechei inhibiting the

growth of fish pathogens as opined by Geraylou,

Vanhove-Maarten, Souffreau, Rurangwa, Buyse

and Ollevier (2014).

The ability of the probiotics to interfere with the

growth of fish pathogens were justified in the pres-

ent study through the antagonistic ability in terms

of their cellular components, which are in agree-

ment with a few previous reports (Abbass, Shari-

fuzzaman & Austin 2010; Sharifuzzaman, Abbass,

Tinsley & Austin 2011). The observed inhibitory

activity cannot be attributed to the acidity in view

of application of the neutralized cellular

CC1FG2 CC1FG4

Bacillus methylotrophicus EU194897.1 CC1HG5

Bacillus subtilis subsp. spizizenii AF074970.1 Bacillus circulans AY724690.1 Bacillus megaterium AB271751.1

Bacillus flexus AB021185.1 Bacillus mycoides AB681413.1 Bacillus anthracis KC020169.1 Bacillus cereus AB592491.1

Bacillus sphaericus gene AB271742.1 Lactobacillus plantarum AB289250.1

Streptococcus agalactiae AB596948.1 Pseudomonas putida AB680572.1

Proteus mirabilis AB626123.1 Klebsiella pneumoniae subsp. ozaenae AF130982.1 Enterobacter cancerogenus Z96078.1 Enterobacter asburiae AB004744.1

Citrobacter freundii AJ233408.1 CC2HG7 Enterobacter hormaechei AJ508302.1

Clostridium botulinum L37585.171674557

10099

100

99

70

99

9499

73

92

42

29

100864539

0.02

Figure 1 Dendogram showing phylogenetic relations of the four most promising probiotic strains, CC1FG2,

CC1FG4, CC1HG5 and CC2HG7, with other closely related type strains retrieved from NCBI GenBank. The GenBank

accession numbers of the reference strains are shown in parentheses. Horizontal bars in the dendogram represent

the branch length. Similarity and homology of the neighbouring sequences have been shown by bootstrap values.

Distance matrix was calculated using Kimura two-parameter model. The scale bar indicates 0.02 substitutions per

nucleotide position. Clostridium botulinum L37585.1 served as an out group.



components (pH 7.2), as indicated by Balcazar

et al. (2008). The extent of the antagonism repre-

sents potential negative interaction between the

resident probiotic bacteria with the tested patho-

genic bacteria. Possible benefit to the host fish can-

not be ruled out as antagonism by the probiotics

will reduce the possibility of establishment of path-

ogenic bacterial population.

Although designating a probiotic organism

might need to possess only one mode of action

(Kesarcodi-Watson, Kaspar, Lategan & Gibson

2008), exploration of other beneficial activities

seemed to be rational to characterize the putative

probiotics. The pathogen inhibitory bacteria

selected in the present study also demonstrated

their ability for extracellular protease, amylase,

lipase, cellulase, phytase and xylanase production.

Enzymes produced by fish intestinal bacteria might

have a considerable role in digestion, especially for

substrates such as cellulose, which only some ani-

mals can digest (Smith 1989). Occurrence of pro-

teolytic, amylolytic and cellulolytic bacteria in the

digestive tracts of tropical freshwater carps has

been recorded (Ghosh et al. 2002a; Saha, Roy, Sen

& Ray 2006; Ray et al. 2010; Ghosh et al. 2010).

Kar et al. (2008) indicated that the enzyme-pro-

ducing gut bacteria might able to utilize carbohy-

drates, such as mannose, xylose, raffinose,

cellobiose and cellulose. Recent observations have

documented that freshwater fish harbour phytase

producing bacteria in their GI tract and assumed

their contribution to overcome the anti-nutritional

effects of plant phytate (Roy, Mondal & Ray 2009;

Khan & Ghosh 2012). Therefore, it may be

hypothesized that the extracellular enzyme-produc-

ing ability of the selected probiotic bacteria might

aid in digestion of feedstuffs and offer additional

benefit to the host fish.

Further, probiotic bacteria should also have the

capacity to tolerate fish GI conditions. The present

study demonstrated competence of the selected iso-

lates to grow/colonize within the gut of C. catla in

terms of their growth potential in mucus and bile

collected from the same species. All of the four

selected gut bacteria grew well in fish mucus, and

intestinal mucus was relatively better growth

media than the skin mucus. Although, bile toler-

ance is considered to be essential for the probiotic

strains, there is no consensus about the accurate

concentration to which a probiotic strain should

be tolerant (Balcazar et al. 2008). Being an agas-

tric fish and slightly alkaline condition therein, the

gut bacteria were isolated and maintained at pH

7.2. Present study revealed that selected probiotic

bacteria could tolerate tested concentrations

(2–20%) of the diluted fish bile juice (pH 5.5–7).Therefore, it might be apprehended that the puta-

tive probiotics characterized in the present report

are likely to survive through the fish GI tract and

colonize. The physiological concentration of bile

was estimated to be approximately 0.4–1.3% in

the fish GI tract (Balcazar et al. 2008). Therefore,

the bile concentration used in the study was com-

paratively high. Similar observations were

recorded by several authors to determine bile sen-

sitivity of the putative probiotic bacteria against

either fish bile (Nikoskelainen et al. 2001; Bur-

bank, LaPatra, Fornshell & Cain 2012) or com-

mercial bile salts (P�erez-S�anchez, Balc�azar, Garcia,

Halaihel, Vendrell, de Blas, Merrifield & Ruiz-Zar-

zuela 2011; Geraylou et al. 2014). Safety of the

host is another prerequisite for any probiotic bac-

terium as suggested elsewhere (Verschuere, Rom-

baut, Sorgeloos & Verstraete 2000). It has already

been established that some strains of B. cereus or

B. subtilis (Pychynski, Malanowska & Kozlowski

1981) are harmful, whereas other strains can be

beneficial as probiotics for animals (Ryan & Ray

2004). In this study, the selected isolates were

evaluated for safety measurement through small-

scale in vivo study and experimental results

revealed that the isolates did not induce any path-

ological signs or mortalities in C. catla.

The use of commercial probiotics in fish is some-

what less effective as the strains used in most of

the commercial preparations are isolated from

non-fish sources and their viability (or survivabil-

ity) within the microenvironment of fish gut at the

required level is doubtful (Moriarty 1996). Hence,

isolation of putative probiotics from the host in

which it has been intended for use is justified.

However, efficiency of the probiotic isolates from

tropical freshwater species is less studied and needs

comprehensive investigation. To the authors’

knowledge, this is the first report pertaining patho-

gen inhibitory property of the cellular components

of Bacillus methylotrophicus (CC1FG2 and CC1FG4),

Bacillus subtilis subsp. spizizenii (CC1HG5) and

Enterobacter hormaechei (CC2HG7) isolated from

the gut of C. catla. Besides, the selected bacteria

could produce extracellular enzymes, were compe-

tent to grow in intestinal condition and safe to the

target species. Detailed characterization of the anti-

pathogenic compounds has not been dealt with in



the present investigation and an appraisal in this

regard will be directed in forthcoming studies. Fur-

thermore, it should be considered that the inhibi-

tion due to such compounds is highly dependent

on the experimental conditions, which are different

in vitro and in vivo (Gatesoupe 1999), and there-

fore, in vivo studies are indispensable with these

autochthonous strains to determine their individ-

ual or combined effects on the host fish to extend

their benefit in the commercial aquaculture.

Acknowledgments

The authors are grateful to the Head, Department

of Zoology, The University of Burdwan, West Ben-

gal, India; The Department of Science and Tech-

nology (FIST programme), New Delhi, India and

The University Grants Commission (UGC-SAP-DRS

programme), New Delhi, India for providing

research facilities. The authors are obliged to Dr.

G. Aditya for rendering help in statistical analyses

of data. Generous gift of some of the pathogenic

strains from Prof. G. Chandra is also thankfully

acknowledged. The first author is grateful to the

DST-INSPIRE programme for awarding the

research fellowship.

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Antagonism against fish pathogens by cellular components and verification of probiotic properties in autochthonous bacteria isolated from the gut of an Indian major carp,Catla catla(Hamilton)