DOI: Dutta et al. Characterization of gut-associated bacteria as probiotics in Labeo rohita POTENTIAL GUT ADHERENT PROBIOTIC BACTERIA ISOLATED FROM ROHU, LABEO ROHITA (ACTINOPTERYGII: CYPRINIFORMES: CYPRINIDAE): CHARACTERISATION, EXO-ENZYME PRODUCTION, PATHOGEN INHIBITION, CELL SURFACE HYDROPHOBICITY, AND BIO-FILM FORMATION Dipanjan DUTTA 1, 2 , Sudeshna BANERJEE 1 , Anjan MUKHERJEE 1 , and Koushik GHOSH 1* 1 Aquaculture Laboratory, Department of Zoology, University of Burdwan, Golapbag, Burdwan, West Bengal, India 2 Post graduate Department of Zoology, Hooghly Mohsin College, Chinsurah, West Bengal, India [The above are only affiliations. No street-address details please] *Correspondence: Dr. K. Ghosh, Aquaculture Laboratory, Department of Zoology, The University of Burdwan, Golapbag, Burdwan 713104, West Bengal, India, phone: +91 9434251606, e- mail: (KG) [email protected], (DD) [email protected], (SB) [email protected], (AM) [email protected]. [Please note that all live links should be removed from e-mail addresses] 1
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· Web viewtract of rohu, Labeo rohita (Hamilton, 1822). Apart from characterization of functional probiotic attributes and bio-safety, the presently reported study utilized in vitro
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DOI:
Dutta et al.
Characterization of gut-associated bacteria as probiotics in Labeo rohita
POTENTIAL GUT ADHERENT PROBIOTIC BACTERIA ISOLATED FROM
and established as a pathogen in a previous study (Mukherjee and Ghosh 2016), was used.
The promising enzyme-producing isolates were tested for pathogen inhibitory activity
against 6 fish pathogens primarily by cross-streaking method (Alippi and Reynaldi 2006) and
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the strains showing antagonistic activity against ≥ 1 studied fish pathogens were further
confirmed by the double-layer method (Dopazo et al. 1988) with minor modification. A clear
zone of inhibition (halo) around growth of the selected gut bacteria indicated antibacterial
activity and the halo (diameter in excess of colony growth) around the colony was presented
as follows; +, low (6–10 mm); ++, moderate (11–20mm); +++, high (21–25 mm); ++++, very
high (≥26 mm).
The bacteriocin-like compound produced by the two promising isolates (LR3H1A and
LR3F3P) was partially characterized after Giri et al. (2012) and Mukherjee et al. (2016) with
minor modification. Following growth in TSB (30°C, 48 h), cell-free supernatant (CFS) was
collected, filter sterilized (0.22 μm; HiMedia, Mumbai, India), and stored at –80°C for further
use as crude bacteriocin (CB). The CB obtained from LR3H1A and LR3F3P were subjected
to heat treatment, pH alteration, and enzyme treatment (α-amylase, trypsin, proteinase K and
lysozyme; 1.0 mg mL–1), following which the treated CB were studied for inhibition of AH by
agar well diffusion method. The CB sample without treatment was served as control in each
case.
Morphological, physiological and biochemical characterization of the bacteria isolates.
Two selected isolates (LR3H1A and LR3F3P) were subjected to morphological,
physiological, and various biochemical tests following standard methods described in the
Bergey’s Manual of Systematic Bacteriology (Bergey and Holt 1994).
Identification of the isolates by 16S rRNA partial gene sequence analysis. LR3H1A and
LR3F3P were further studied by 16S rRNA partial gene sequence analyses following the
methods described in Dutta et al. (2015). The 16S rRNA gene fragments were amplified by
polymerase chain reaction (PCR) using universal primers, viz. 27f (5′-
AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-GGTTACCTTGTTACGACTT-3′) (Lane
1991). Sequencing products were determined on an automated DNA sequencer (Applied
BioSystems 3730XL, USA), edited with the BioEdit Sequence Alignment Editor (Version
7.2.5), aligned and analysed to find out the closest homolog using National Centre for
Biotechnology Information (NCBI) GenBank and Ribosomal Database Project (RDP)
databases. Sequences were deposited to the NCBI GenBank and accession numbers were
obtained. A phylogenetic tree was constructed using MEGA 7 software following the
Minimum Evolution method.
Ability to tolerate gastrointestinal condition. The two selected bacteria, LR3H1A and
LR3F3P were assayed for their ability to grow in fish mucus and tolerance toward diluted bile
juice following the method described by Dutta and Ghosh (2015).
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Cell surface hydrophobicity assays. A total of five assays were performed:
Spontaneous aggregation assay. An overnight grown broth culture of LR3H1A and
LR3F3P in TSB was placed on a clean glass slide and rotated manually. Clumping of cells
inferred a positive result, whereas a smooth turbid suspension indicated a negative result
(Mattos-Guaraldi et al. 1999).
Salt aggregation assay. Salt aggregation assay was performed after Krepsky et al.
(2003) with slight modification. Different grades of ammonium sulphate (0.007M to 4M)
were mixed with a suspension of LR3H1A and LR3F3P (100 µL) in a watch glass. The
lowest grade of ammonium sulphate giving visible bacterial clumping was scored as salt
aggregation index. Bacterial suspension with a salt aggregation index < 2M was considered as
positive.
Auto-aggregation assay. Auto-aggregation assays were performed according to Kos et
al. (2003) with minor modifications. The cells of overnight grown broth cultures of LR3H1A
and LR3F3P (resuspended in PBS) were mixed using a vortex (10 s) and incubated (5 h) at
25°C. The absorbance (A600) was recorded for a period of 4 h at an interval of 1h. The
autoaggregation (AA; %) ability was expressed as follows:
AA = 1 – 100 · (At · A0–1)
where, At represents the absorbance at time t = 1, 2, 3, and 4 h, and A0 represents the
absorbance at t = 0.
Co-aggregation assay. Co-aggregation assays were performed according to Jena et al.
(2013) with slight alterations. Equal volume (1 : 1) of each probiotic and pathogenic
suspension (AS, PF, PP, BM, AH) were thoroughly mixed using a vortex (10 s). The tubes
were incubated (5 h) at 25°C. The co‐aggregation (CA; %) ability was expressed as follows:
CA = 100 · (A0 – At) · A0–1
where, A0 represents OD600 of a bacterial mixture at t = 0, and At represents the OD600 of a
bacterial mixture after 5 h of incubation.
Microbial adherence to hydrocarbon (MATH). MATH was performed according to
Klayraung et al. (2008) with modifications. Solvents used in the present study were n-
hexadecane (apolar, n-alkane), xylene (apolar), chloroform (monopolar, Lewis-acid), and
ethyl acetate (monopolar, Lewis-base). Broth cultures of LR3H1A and LR3F3P (resuspended
in PBS) were grown overnight and OD600 was measured (A1). Respective solvents (1 mL) were
added separately to each cell suspension, incubated at 30°C for 30 min and OD600 measured
against a solvent-extracted PBS blank (A2). Percentage of adhesion (Adhs; %) was expressed as
follows:
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Adhs = 100 · (1 – A2 · A1–1)
Ability to form bio-film. Two assays were performed:
Cell adherence to glass and polystyrene surfaces. Preliminary measurement of cell
adherence to glass and polystyrene tubes were compared for both strains. Broth cultures of
LR3H1A and LR3F3P (108 CFU mL–1) were incubated overnight at 30°C under static
condition in glass and polystyrene tubes (5 mL). OD600 of the homogeneously mixed culture
was measured (A) in both the tubes. The culture was centrifuged at 5000 g for 15 min, the
supernatant was collected and OD600 was measured (A0). The process was repeated for three
days by inoculating fresh broth with decanted supernatant. Percentage of adhesion (Adhr; %)
was expressed as follows:
Adhr = 100 · (A – A0 · A–1)·
Semi-quantitative adherence assay. Bio-film production by LR3H1A and LR3F3P was
determined using a semi-quantitative adherence assay on polystyrene made 96-well tissue
culture plates following Chaieb et al. (2007). OD570 of the adherent and stained bacteria were
measured. OD570 < 0.1 was noted as non bio-film producer and OD570 > 1.0 was considered as
strong bio-film producer.
Safety evaluation. The strains LR3H1A and LR3F3P were injected intra-intraperitoneally
into rohu fingerlings (20 for each strain; mean weight: 16.5 ± 1.25 g) and was observed for
mortality or onset of disease symptoms (Dutta and Ghosh 2015).
Statistical analysis. Statistical analyses of the quantitative enzyme activity, hydrophobicity
assays, and partial characterization of bacteriocin were performed by the one-way analysis of
variance (ANOVA) followed by Tukey’s test according to Zar (2010) using SPSS Version 19
software (Gray and Kinnear 2011).
RESULTS
Isolation of gut bacteria and determination of extracellular enzyme-producing capacity.
Amylase, protease, lipase, cellulase, phytase, and xylanase producing autochthonous bacteria
populations were detected in the proximal (PI) and distal (DI) segments of the GI tract of rohu
(Fig. 1). Heterotrophic and diverse extracellular enzyme-producing bacterial populations were
predominantly high in the DI region. While considering different extracellular enzyme-
producing bacteria on a comparative scale, the occurrence of amylolytic bacteria was the
highest (LVC = 5.65) followed by lipolytic bacteria (LVC = 5.34), both in the DI segment;
whereas, xylan-degrading bacteria populations were the lowest in the PI and DI. Seven
extracellular enzyme-producing strains (4 from PI and 3 from DI) were primarily selected on
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account of qualitative enzyme activity data presented as scores (Table 1), maximum and
minimum scores being 28 and 20, respectively. Further, results of the quantitative enzyme
assay revealed significant differences in the enzyme activities among the primarily selected
bacterial isolates (Table 2).
The highest values for amylolytic (300.60 ± 6.04 U), proteolytic (82.38 ± 2.14 U) and
phytase (378.51 ± 6.22 U) activities were recorded by strain LR3F3P. While, strain LR3H1A
revealed maximum cellulase (72.41 ± 2.31 U), xylanase (33.68 ± 1.21 U) and lipase (5.93 ±
0.28 U) activities. Overall examinations of the six different extracellular enzyme activities
revealed that strains LR3H1A and LR3F3P (qualitative activity score was 28 and 27,
respectively) were the most efficient among the 7 primarily selected bacteria strains.
Determination of antagonistic activity against pathogenic bacteria. To verify pathogen
inhibitory activity, the primarily selected 7 extracellular enzyme-producing bacterial isolates
were further screened against 6 fish pathogens. Out of the 7 isolates, 3 strains (2 from PI and
1 from DI) inhibited at least one of the tested fish pathogens through cross-streaking method.
None of the exoenzyme-producing gut isolates was antagonistic against Bacillus mycoides.
Pathogen inhibitory activity of these 3 isolates was further assessed by double layer method
and the zone of inhibition (halo) produced by the gut isolates were depicted in Table 3. In
consequence of the maximum extracellular enzyme-producing capacities; strains LR3H1A
and LR3F3P were antagonistic against 3 out of the 6 tested fish pathogens.
Partial characterization of bacteriocin-like compound. The effect of different treatments
on the inhibitory activity of the antagonistic compound produced by LR3H1A and LR3F3P
against Aeromonas hydrophila MTCC-1739 has been depicted in Fig. 2. Inhibitory activity of
the antagonistic compound reduced significantly (P < 0.05) with increasing temperature and
was completely inactivated at temperatures above 90°C. The antibacterial activity was lost
when the antagonistic compound was treated with proteolytic enzymes (proteinase K and
trypsin), however, remained active under the action of lysozyme and α-amylase. Therefore,
the study revealed that the antagonistic compound was proteinaceous in nature and
bacteriocin like. The bacteriocin-like compound produced by both the bacteria remained
active following the exposure to a wide range of pH conditions (pH 3−9).
Morphological, physiological, and biochemical characterization of the selected isolates.
Both strains were Gram-positive motile rods, catalase positive and capable of utilizing citrate,
ribose, xylose, galactose, trehalose, starch, glycogen, etc., but the strains differed in some
characteristics. LR3F3P revealed positive result for tryptophan deaminase, indole, and nitrate
production. It was capable of utilizing a number of carbon sources and amino acids like
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mannitol, inositol, sorbitol, arginine, lysine, ornithine, and others. However, LR3F3P,
LR3H1A could utilize D-melibiose as a carbon source (Table 4).
Genotypic identification of the selected isolates. Based on the nucleotide homology and
phylogenetic analysis of the 16S rRNA partial gene sequences by nucleotide blast in the
NCBI GenBank and RDP databases, the putative probiotic strain LR3H1A was identified as
B. subtilis subsp. spizizenii (GenBank accession No. KF623286) showing 99% similarity to
the type strain B. subtilis subsp. spizizenii (AF074970.1). Isolate, LR3F3P showed 99%
similarity with the type strain B. tequilensis (HQ223107.1) and was identified as B.
tequilensis (GenBank accession No. KF623287). Phylogenetic relation of the two identified
bacterial isolates with other closely related type strains retrieved from the RDP database is
presented in the dendrogram (Fig. 3).
Ability to tolerate gastrointestinal condition. The strains, LR3H1A and LR3F3P, grew well
in mucus collected from rohu (data not shown) and were capable to withstand diluted bile
juice up to a concentration of 11% and 12%, respectively (data not shown).
Cell surface hydrophobicity assays. Both the isolates (LR3H1A and LR3F3P) showed
moderate clumping in spontaneous aggregation assay indicating hydrophobicity of the strains.
In the salt aggregation test, LR3H1A and LR3F3P showed clumping at 1M and 1.25M
ammonium sulphate, respectively. Being eventually lesser than the threshold limit of 2M,
both the strains indicated moderate hydrophobic cell surface. The auto-aggregation ability
measured over a period of 4 h exhibited a moderate to strong auto-aggregating attribute of the
strains. The ability of auto-aggregation increased with increasing incubation period (Fig. 4a).
While comparing auto‐aggregation ability (4 h), strain LR3F3P revealed better auto‐aggregation ability (49.33 ± 1.35%) than strain LR3H1A (32.45 ± 1.43%). This result could
indicate a potential capability of the strains to adhere to epithelial cells and mucosal surfaces.
Both bacteria strains were able to co‐aggregate with the tested pathogens at varied levels (Fig.
4b). Isolate LR3F3P showed the highest co-aggregation percentage with A. salmonicida
(36.08 ± 1.31%) and the least with B. mycoides (20.37 ± 1.05%). On the other hand, isolate
LR3H1A illustrated strong co‐aggregation with A. hydrophila (23.75 ± 1.29%) but less with
P. fluorescens (11.55 ± 0.98%).
Cell surface properties revealed through MATH measurement indicated that both the
putative probiotic strains were more of hydrophobic (than hydrophilic) with strong adhesion
to xylene, which is an apolar solvent (Fig. 4c). Bacterial adhesion to chloroform and ethyl
acetate was evaluated to assess the Lewis acid-base characteristics of cell surfaces. Both the
strains showed a stronger affinity towards chloroform, which is an acidic solvent and electron
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acceptor, than the ethyl acetate, which is a basic solvent and electron donor. Thus, the results
might indicate that metabolically the cells are better electron donor and weak electron
acceptor.
Ability to form bio-film. Both strains were adherent to polystyrene and glass surfaces
following 24 h of interaction, however, adhesion increased with increasing time of interaction
(Fig. 4d).
On a comparative scale, both the strains exhibited a stronger affinity (adherence)
towards polystyrene than the glass surface. Further, the strains were noticed to be moderate