Aquaculture 548 (2022) 737597
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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture
Optimization of the multispecies probiotic combination with
N-acylhomoserine lactone-degrading ability for increased disease resistance
of Carassius auratus using response surface methodology
Divya Vadassery Haridas a, C.G. Joshy b, Devika Pillai a, *
a
b
Department of Aquatic Animal Health Management, Kerala University of Fisheries and Ocean Studies, Cochin 682 506, Kerala, India
ICAR-Central Institute of Fisheries Technology, Matsyapuri, Cochin 682 029, Kerala, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
N-acyl-homoserine lactones
Multi-species probiotic
Quorum sensing
Quorum quenching
Aquaculture
Response surface methodology
In the present study, two N-acyl homoserine lactone degrading bacteria (Lactobacillus plantarum QQ8 and
Lactobacillus casie QQ10) isolated from the gastrointestinal tract of Oreochromis niloticus were tested for their
probiotic effect on goldfish. The isolates were able to degrade short chain synthetic C6-HSL in vitro and hampered
violacein production by Chromobacterium violaceum. They were susceptible to all the five antibiotics tried. The
isolates grew well at pH 3.0–7.0, were resistant to high level of bile salts (0–0.9%) and 0.5% of phenol. They also
exhibited high degree of auto-aggregation and co-aggregation, confirming that they possessed good probiotic
attributes. Disease resistance in goldfish against Aeromonas hydrophila infection was assessed with 28 different
combinations of probiotic mixtures of the four bacterial strains:L. plantarum QQ8, L. casei QQ10, Enterococcus
faecium QQ12 and Bacillus thuringiensis QQ17 (QQ12 and QQ17 were previously isolated in our laboratory) by
using the statistical approach of response surface methodology (RSM). The ability of goldfish to resist
A. hydrophila infection was higher in probiotic combination fed treatments than that fed with commercial feed
alone. The highest survival was observed in gold fish fed diet incorporated with the combination dose of 103
CFU/g feed of L. plantarum QQ8, 102 CFU/g feed of L. casei QQ10, 102 CFU/g feed of E. faecium QQ12 and 103
CFU/g feed of B. thuringiensis QQ17. The corresponding desirability score was almost 1. Goldfish showed 91.7%
survival when challenged with A. hydrophila. This study demonstrated the potential of multi-species quorum
quenching probiotics as feed additive in aquaculture to control bacterial diseases and use of response surface
methodology in determining the optimum combination of probiotic mixture.
1. Introduction
In aquaculture, intensification of rearing system to meet the growing
demands for human consumption has led to the sudden occurrence of
bacterial diseases, especially, those generated by Gram-negative bacte­
ria. They are regarded as one of the major cause of mortalities in
aquaculture (Novriadi, 2016). Among the Gram-negative bacteria,
Aeromonas species are common enteric pathogens causing motile aer­
omonas septicemia or ulcer disease in fish (Bullock et al., 1971; Egusa,
1978). This disease troubles plenty of tropical or ornamental fish. The
control of bacterial diseases in aquaculture is very often achieved
through application of antibiotic. However, improper use of antibiotics
can lead to the development of antibiotic resistance in pathogenic bac­
teria (Komolafe, 2003). Unfortunately, due to the rising rate of antibiotic
resistance, the discovery of new antibiotics has critically declined in the
last several decades (Chen et al., 2013). Therefore, novel alternatives are
needed for prophylaxis and treatment of diseases, in order to maintain
sustainable development of aquaculture industry. One such novel
approach is the administration of beneficial bacteria disrupting the
pathogenic bacterial communication systems (quorum sensing) con­
trolling the expression of virulence factors rather than their elemental
structures or processes.
It is evident that in most of the Gram-negative bacteria, pathoge­
nicity relies on the quorum sensing (QS) process, where virulent gene
expression is mediated by extracellular signaling molecules called Nacyl homoserine lactones (AHL) (Federle and Bassler, 2003). Quorum
Quenching (QQ) is the mechanism of intercepting QS by signal
degrading enzymes recognized in many bacterial groups, such as AHL
* Corresponding author.
E-mail addresses: divyavhmbt@gmail.com (D. Vadassery Haridas), cgjoshy@gmail.com (C.G. Joshy), dr@kufos.ac.in (D. Pillai).
https://doi.org/10.1016/j.aquaculture.2021.737597
Received 27 November 2020; Received in revised form 23 April 2021; Accepted 13 October 2021
Available online 20 October 2021
0044-8486/© 2021 Elsevier B.V. All rights reserved.
D. Vadassery Haridas et al.
Aquaculture 548 (2022) 737597
lactonase, AHL acylase, and AHL oxidoreductase and has been suggested
as a potential biocontrol approach to attenuate bacterial pathogens
(Dong et al., 2000), which could serve as a substitute to conventional
antibiotic control of bacterial infections in aquatic systems (Cam et al.,
2009; Defoirdt et al., 2007; Dong et al., 2001; Lin et al., 2003; Zhang
et al., 2002). As a well identified alternative to antibiotics, the appli­
cation of probiotics too is gaining acceptance for the control of patho­
genic bacteria in aquaculture. Possible modes of probiotic action to
remove pathogens in aquatic animals include: competitive exclusion of
pathogenic bacteria by removal of available bacterial receptor sites,
competitive consumption of essential nutrients, synthesis and secretion
of inhibitory compounds and antimicrobial substances, improving water
quality, improvement of the immune system, production of essential
nutrients and enzymatic contribution to digestion (Adams, 2010;
Lakshmi et al., 2013; Zhou et al., 2010). Probiotics have been used in
aquaculture during the last three decades. In this circumstance, use of
AHL degrading bacteria which can at the same time function as probiotic
would be a distinctive approach to control aquatic pathogens effectively
and to help the host in a positive way. Recently, various research works
have been reported on AHL degrading bacteria isolated from digestive
tract of aquatic animals that are capable of utilizing AHL molecules of
aquatic pathogenic bacteria as a food source (Cam et al., 2009; Chu
et al., 2014; Nhan et al., 2010; Ramesh et al., 2014; Vinoj et al., 2014). It
has also been demonstrated that probiotic bacteria such as Enterococcus
durans, Enterococcus faecium and Bacillus thuringiensis degrade the AHL
molecules of pathogenic bacteria by enzymatic action (Boopathi et al.,
2017; Chu et al., 2010; Vadassery and Pillai, 2019, 2020).
In the present study we investigated the quorum quenching attri­
butes and probiotic properties of two bacterial isolates (Lactobacillus
plantarum QQ8 and Lactobacillus casei QQ10) from gastrointestinal tract
of Oreochromis niloticus. It also explored the use of multi-species pro­
biotic to reduce Aeromonas hydrophila infection in goldfish Carassius
auratus. The quorum quenching efficiency of the individual as well as
mixture of QQ isolates was also ascertained. In the present work, we
used Response Surface Methodology (RSM) to examine the effect of
various probiotic combinations of four AHL degrading bacteria:
L. plantarum QQ8, L. casei QQ10, E. faecium QQ12 and B. thuringiensis
QQ17 [QQ12 and QQ17 were previously isolated in our laboratory and
tested for their AHL degrading ability and probiotic properties individ­
ually (Vadassery and Pillai, 2019, 2020)] on the disease resistance of
goldfish C. auratus. RSM is a statistical method, applied for the optimi­
zation of different input variables for either maximizing or minimizing
the specific response variables. It is a powerful technique for testing
multiple-process variables because fewer experimental trials are needed
as compared with the study of one variable at a time. The goal of this
study was to choose the best probiotic combination of different micro­
bial biomass of L. plantarum QQ8, L. casei QQ10, E. faecium QQ12 and
B. thuringiensis QQ17 for maximal survival of goldfish against
A. hydrophila infection.
grown in LB medium at 37 ◦ C, served as negative control in AHLinactivation assay. All media used for AHLs assay were buffered with
50 mmolL-1 3-[N-morpholino] propane sulfonic acid (MOPS) to pH 6.8,
to prevent spontaneous degradation of AHLs.
2.2. Isolation and identification of quorum quenching bacteria from
O. niloticus (tilapia) gut
O. niloticus were collected from aquaculture farms in Ernakulum
district, Kerala, India. The fish were killed with an overdose of clove oil
(200 mg l− 1) and dissected aseptically. The gastrointestinal tract was
removed as quickly as possible anda gut homogenate was prepared in
sterile physiological saline [(pH 7.4) 0.85% NaCl]. Samples were then
enriched in minimal medium (KG medium) with AHL as the sole source
of carbon and nitrogen. 100 μl of the homogenate was inoculated into
100 ml flask containing 10 ml of KG medium (pH 6.8) with 500 μg l− 1 of
C6-HSL, as previously described (Chan et al., 2009) and incubated at
28 ◦ C. After 24 h, 1 ml of culture was transferred to fresh C6-HSL con­
taining KG medium for enrichment culturing. At the third time enrich­
ment cycle, a diluted suspension was plated onto LB agar. After 48 h of
incubation, bacteria with different colony morphology were picked and
streaked on LB agar. Pure colonies were obtained by repeated streaking
on LB agar. The selected bacteria were identified following Bergey’s
Manual of Systematic Bacteriology (Ludwig et al., 2009). Species level
identification was carried out by 16S rDNA sequencing (SciGenom Labs,
India) using universal primers 27F and 1492R and analyzed using NCBI
nucleotide database. Antibiotic susceptibility of the isolates was checked
by disc diffusion method following the guidelines of the Clinical and
Laboratory Standard Institute (CLSI, 2017). The antibiotic discs used in
this test included ampicillin (10 μg), erythromycin (15 μg), penicillin G
(10 U), vancomycin (30 μg), and tetracycline (30 μg).
2.3. Bile salt and acid tolerance
The isolates obtained from the enrichment culture (QQ8 and QQ10)
were tested for bile salt tolerance and survival in acidic condition. Each
bacterial strain was grown overnight in MRS media and 0.1 ml of each
culture suspension (absorbance was adjusted to 0.02) was inoculated
into tubes containing 10 ml of autoclaved MRS media with 0.3%, 0.6%
and 0.9% bile salt (Himedia, India). Broth with 0% bile concentration
served as a control. The tubes were incubated at 30 ◦ C for 18 h. Sub­
sequently, 0.1 ml was pipetted out from each of the MRS broth and 10fold serial dilutions were made for plating. All the plates were incubated
at 30 ◦ C for 18 h. Bile tolerance was determined by comparing the viable
cell counts on MRS agar plates with and without bile salt. To determine
acidic tolerance of QQ8 and QQ10, 0.1 ml of actively grown overnight
culture of each isolate at 30 ◦ C in MRS medium was transferred to
autoclaved MRS broth adjusted to pH 1–7 with HCl (Sigma, India).,
which were then incubated at 30 ◦ C for 18 h, followed by plating on MRS
agar plates, as described above. Acid tolerance was determined by
comparing the viable cell counts on the MRS agar plates with acidic pH
and normal pH. Both assays were carried out in triplicate.
2. Materials and methods
2.1. Bacterial strains and growth conditions
2.4. Phenol tolerance assay
The previously isolated E. faecium QQ12 and B. thuringiensis QQ17
(Vadassery and Pillai, 2019, 2020) were grown in Luria Bertani (LB)
medium at 30 ◦ C. The mutant strain Chromobacterium violaceum CV026
was used as biosensor to find out the presence of exogenous C6-HSL. It
was purchased from Microbial Culture Collection (MCC), NCCS, Pune,
India. CV026 cannot synthesize AHL, but it can detect and respond to
exogenous AHLs with acyl chain of four to eight carbons, by production
of the purple coloured violacein pigment. It was grown in LB medium at
28 ◦ C supplemented with 50 μg ml− 1 of kanamycin. The target fish
pathogen A. hydrophila used in this study was provided by the National
Bureau of Fish Genetic Resources (ICAR, Kochi, India). It was grown in
LB broth (pH 7.2 ± 0.2) at 30 ◦ C overnight. Escherichia coli DH5α, also
To check the phenol tolerance, actively growing overnight cultures
of QQ8 and QQ10 were inoculated into MRS media with concentration
of 0.2% and 0.5% phenol or without phenol. Cell growth of the isolates
was evaluated after 18 h of incubation at 30 ◦ C, by measurement of
absorbance at 600 nm. The assay was carried out in triplicate.
2.5. Auto-aggregation and Co-aggregation assays
To evaluate the probiotic potential of QQ8 and QQ10, autoaggregation and co-aggregation rate were measured according to Del
Re et al. (2000) with some modifications. Isolates were grown for 18 h at
2
D. Vadassery Haridas et al.
Aquaculture 548 (2022) 737597
30 ◦ C in MRS media. The cells of each isolate were harvested by
centrifugation at 2800g for 15 min at 4 ◦ C, washed twice with PBS (pH
7.2) and resuspended in the same buffer. Initial absorbance (A600 nm)
was adjusted to 0.2. Cell suspensions (5 ml) were mixed by vortexing for
10 s and the same suspensions were left to rest for 5 h at room tem­
perature without vortexing. Auto-aggregation of each cell suspension
was determined by taking 0.1 ml of the upper suspension at every 1 h
interval to another tube with 4.9 ml of PBS and the absorbance of sus­
pension at 600 nm was recorded. Cell auto-aggregation was measured
by decrease in absorbance and auto-aggregation percentage is demon­
strated as: 1-(At/A0) X 100, where At represents the absorbance at time t
= 1, 2, 3, 4 or 5 h and A0 the absorbance at t = 0.
The procedure for making the cell suspension for co-aggregation
assay was the same as that for auto-aggregation assay. QQ8 and QQ10
prepared as mentioned above were mixed with equal volume (2 ml) of
the culture of fish pathogen A. hydrophila and incubated at room tem­
perature without agitation. In control tubes, 4 ml of each bacterial
suspension alone was added. After 5 h of incubation, the absorbance (A)
at 600 nm of the suspensions was measured. Co-aggregation percentage
was calculated using the equation of Handley et al. (1987). Coaggregation % = [(Apathog + AQQ)/2 - (Amix) / (Apathog + AQQ)/2] X
100, where Apathog and AQQ constitute the absorbance in the tubes con­
taining solely the pathogen or the quorum quenching bacteria (control
tubes) respectively, and Amix represents the absorbance of the mixture.
Auto-aggregation and co-aggregation assays were performed in
triplicate.
Table 1
Survival percentages of Carassius auratus (fed with different concentrations of
probiotic diet) two weeks after the experimental infection with Aeromonas
hydrophila by intraperitoneal injection.
Isolates with different cell densities (CFU/g feed)
2.6. Quantification of AHLs degradation by microtiter plate assay
AHL degradation potential of each of the QQ isolate (QQ 8, QQ10,
QQ12 and QQ17) was quantified individually as previously reported by
Zhu et al. (2011) with some modifications. The quorum quenching ef­
ficiency of the mixture of QQ isolates was also checked. It was performed
using a microtiter plate assay by measuring the intensity of violacein
production by CV026 in the presence of AHL degrading strains. Actively
grown biosensor CV026 was inoculated to appropriate growth medium
containing overnight culture of each of the AHL degrading bacteria
supplemented with 10 mM C6-HSL.The procedure for making the AHL
degrading media for the mixture of QQ isolates was the same as that for
individual isolate. The initial bacterial cell density was taken as 1 × 104
CFU in both individual and combination AHL degradation media.
Briefly, the mixtures were incubated at 30 ◦ C for 24 h in shaking incu­
bator (120 RPM). Then, 1 ml of each of the reaction mixtures was
transferred to microcentrifuge tube containing 1 ml of dimethyl sulph­
oxide (DMSO) and centrifuged at 10000 rpm for 5 min to solubilize
violacein and to precipitate the cells. 200 μl of violacein containing
supernatants were added to 96-well microplates (Tarsons) and the
absorbance was measured in a microplate reader (Biorad, USA) at a
wavelength of 585 nm. Three replicates were performed for each
quorum quenching isolate. The results were compared with negative
control (DH5α) result. The percentage of violacein inhibition (AHL
degradation) was calculated by the formula: - (Control OD585 nm – Test
OD585 nm/control OD585 nm) × 100.
QQ8
QQ10
QQ12
QQ17
Run
(X1)
(X2)
(X3)
(X4)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
0
101
0
1010
0
0
107
0
103
0
103
101
103
103
0
103
107
0
107
101
103
0
0
107
0
1010
0
103
103
107
103
0
1010
103
0
107
0
0
103
101
0
103
0
103
0
0
101
101
0
1010
0
103
107
0
0
107
103
101
0
0
0
107
0
0
0
1010
0
107
103
103
103
104
103
1010
101
101
107
0
0
0
103
0
107
0
Survival %
104
101
107
0
0
0
103
103
107
0
104
101
104
104
107
0
0
0
101
107
0
0
1010
0
0
0
103
0
83.3±0
87.5±5.9
66.7±0
79.15±5.9
66.7±0
70.85±5.9
62.5±5.9
58.3±0
58.3±0
83.3±0
87.5±5.9
83.3±0
91.7±0
91.7±0
58.3±0
87.5±5.9
45.8±5.9
83.3±0
83.3±0
75±0
54.15±5.9
66.7±0
79.15±5.9
41.6±0
50±0
79.15±5.9
58.3±0
50±0
Note: Values are mean ± SD of triplicate observations.
2.7.2. Maintenance of experimental fish
Fingerlings of goldfish C. auratus (Linnaeus, 1758) of uniform size
were initially acclimatized in fibre reinforced plastic tanks of 300 l ca­
pacity for three weeks before starting the experiment. The fish were
healthy, exhibited no signs of disease (confirmed through the active
swimming movement, examination of gills, fins and skin). The pathogen
free status of the fish was confirmed by determination of total viable
aerobic count by standard plate count method (Maturin and Peeler,
2001). During this period, the fish were fed with a commercial fish feed
twice daily. All tanks were provided with proper aeration (Dissolved
oxygen level was maintained at 5–7 ppm) and water temperature was
maintained at 26 ± 1 ◦ C. pH was recorded at regular intervals and
maintained at 7.2 ± 0.1. After the acclimatization, 28 groups of 12
goldfish each (each group maintained in triplicate) were introduced into
84 experimental glass tanks of 50 l capacity. Control group in triplicate
was also maintained.
2.7.3. Safety of the QQ8 and QQ10
The pathogenicity of QQ8 and QQ10 was tested before preparing
probiotic feed (Safety of QQ12 and QQ17 was previously tested)
(Vadassery and Pillai, 2019, 2020). Two groups of six goldfish each
(3.34–4.32 g weight and 85.35–94.40 mm length), were challenged with
0.1 ml of PBS containing 1.0 × 1010 cells of each QQ bacteria by
intraperitoneal injection. Each group was maintained in triplicate. The
control group fish received 0.1 ml of PBS alone. Fish were observed for
mortality for seven days. During this period behaviour of fish was
recorded daily.
2.7. Optimization of the microbial probiotic combination for the disease
resistance in goldfish against A. hydrophila infection by response surface
methodology (RSM)
2.7.1. Experimental design
The present work was performed to optimize the best probiotic
combination of four different AHL degrading bacteria; QQ8 (x1), QQ10
(x2), QQ12 (x3) and QQ17 (x4) to increase the disease resistance in
goldfish C. auratus against A. hydrophila infection. In this regard, a
randomized simple lattice mixture response surface design with 28 runs
was formulated. The combination of experimental design is given in
Table 1.
2.7.4. Preparation of probiotic feed and feeding trial
According to the 28 runs of batch experimental setup of response
surface methodology, 28 probiotic combinations were prepared (The
feed combinations are mentioned in Table 1) using different
3
D. Vadassery Haridas et al.
Aquaculture 548 (2022) 737597
concentrations of AHL degrading bacteria QQ8, QQ10, QQ12 and QQ17
and incorporated to the commercial feed using a binder (Brand: Aqua
one, Salem Microbes Private limited, India) at a rate of 1 mL 10 g− 1 feed.
Binder alone was added in control feeds. After proper mixing of the
ingredients, the feeds were air dried and stored in screw capped glass
bottles at room temperature until used. To ensure a required probiotic
level in the supplemented feed, new probiotic diets were made on a
weekly basis. Feeding was done two times daily at the rate of 3% of the
body weight of C. auratus for 30 days. The fish of all groups were fed
with respective feed in triplicate. Continuous aeration and water flow
were maintained in all glass tanks. During the study period, activity and
behaviour of the fish were monitored and recorded daily.
2.8. Statistical analysis
All the experiments were performed in triplicate and the results were
expressed as mean ± standard deviation (SD) of triplicates. Data were
statistically processed by one way ANOVA using SPSS (Version 21.0).
Statistically significant differences were defined at p ≤ 0.01.
3. Results
3.1. Isolation and identification of quorum quenching bacteria
Two bacterial isolates (QQ8 and QQ10) showing AHL degrading
activity in the KG medium containing C6-HSL were screened and char­
acterized at the physiological, biochemical and morphology levels.
Based on biochemical properties, the strains showed close resemblance
to Lactobacillus spp. To further identify the strains, 16S rDNA sequencing
was carried out. Results showed the QQ8 and QQ10 shared 100% ho­
mology with L. plantarum and L. casei (GenBank accession numbers
KJ22782 and MK643164) respectively. The isolates were susceptible to
five antibiotics tested (ampicillin, erythromycin, penicillin G, tetracy­
cline and vancomycin) (Table 2).
2.7.5. Bacterial challenge study
After 30 days of feeding, the fish were challenged with A. hydrophila.
Before conducting the challenge study, the infectious dose of
A. hydrophila was selected by 50% lethal dose (LD50) determination
(based on the study carried out previously in our lab) (Vadassery and
Pillai, 2019, 2020). Controls and probiotic fed fish (28 groups; 12
numbers in each group, maintained in triplicate) were challenged via
intraperitoneal injection with 0.1 ml of 1 × 106 (LD50) cells of
A. hydrophila. External signs of infection, behavioural abnormalities and
mortalities, if any, were recorded for two weeks. Dead fish were
removed immediately for bacteriological examination. Bacterial isola­
tion was carried out from hemorrhagic and ulcerative lesions and
visceral organs. Liver, head, kidney and spleen tissues from dead fish
were plated on TSB agar plates to detect the presence of bacteria.
Identification of re-isolated A. hydrophila was done by PCR amplification
and sequencing of 16S rDNA (SciGenom Labs, India) using universal
primers 27F and 1492R and analyzed using NCBI nucleotide database.
3.2. Screening of probiotic activities
L. plantarum QQ8 and L. casei QQ10 grew successfully in all tested
concentrations of bile (0–0.9%) after 18 h of incubation (Fig. 1). There
were no significant differences in log10 CFU/ ml between control
(without addition of bile salt, pH 7) and QQ isolates with different
concentrations of bile salt after 18 h of incubation (Fig. 1). This data
suggests that L. plantarum QQ8 and L. casei QQ10 are resistant to high
bile salt concentration. pH tolerance studies showed that L. plantarum
QQ8 and L. casei QQ10 grew at pH 3 and above but did not grow in
conditions less than pH 3 (Fig. 2). The two isolates grew well at 0–0.5%
of phenol in growth media (Fig. 3).
2.7.6. Development of statistical models
Cubic mixture response surface models were fitted to the experi­
mental data and the same was used to predict the response variable
survival rate as a function of input variables. The mathematical form of
cubic model is given in Eq. (1). The models were fitted to the experi­
mental data using Design Expert 7.1.5.
Cubic model:
q
∑
Y=
q
∑
∑
βij xi xj +
βi x i +
i=1
i<j=2
∑ ∑ ∑q
β xi xj xk + e⋯
i<j<k ijk
3.3. Auto-aggregation and Co-aggregation
The aggregation rate of L. plantarum QQ8 and L. casei QQ10 was
measured every hour for 5 h. The results showed that the two QQ iso­
lates had good auto-aggregation property and aggregation values
increased with time (Fig. 4). L. plantarum QQ8 exhibited highest autoaggregation rate (77.73 ± 0.13%) followed by L. casei QQ10 (66.73 ±
0.04%) (Fig. 4).They also exhibited very good co-aggregation ability
after 5 h of incubation with A. hydrophila. L. plantarum QQ 8 showed the
highest co-aggregation and 39.61 ± 0.23% of QQ8 co-aggregated with
A. hydrophlila (Fig. 5). The co-aggregation rate showed by L. casei QQ10
was 29.4 ± 0.18% (Fig. 5).
(1)
where Y is survival rate and xi, i = 1, 2, 3, 4 [QQ8 (x1), QQ10 (x2),
QQ12 (x3) and QQ17 (x4)] is input variables, βi is linear regression co­
efficient, βij is quadratic regression coefficient, βijk is cubic regression
coefficient and ‘e’ is the error term. The regression coefficients were
estimated by ordinary least squares (OLS) method. Desirability function
has been formulated and desirability score has been computed for
maximizing the survival rate. The goodness of fit of the model was
assessed by coefficient of determination (R2) and root mean square error
(RMSE). The fitted model with highest R2 and lowest RMSE values was
selected for predicting the response variables.
Table 2
Antibiotic resistances of Lactobacillus plantarum QQ8 and Lactobacillus casei
QQ10.
Zone of growth inhibition diameter (mm)
2.7.7. Validation of the optimized condition
On the basis of the results obtained from statistical analysis, the
optimum probiotic combination was derived. A validation study was
carried out at optimum combination of input variables in triplicate. In
this combination, probiotic feed was prepared and feeding was done
with respective feed two times daily to goldfish in glass tanks (12
goldfish in each tank) for 30 days. During this period, dissolved oxygen,
temperature and pH were maintained at 5–7 ppm, 26 ± 1 ◦ C and 7.2 ±
0.1 respectively. Then, probiotic fed fish were challenged with 0.1 mL of
1 × 106 cells of A. hydrophila and were observed for external signs of
infection, behavioural abnormalities and mortalities, if any, for two
weeks.
Isolates
L. plantarum
L. casei
AM
E
P
TE
28 ± 0.26
24 ± 0.35
26 ± 0.39
25 ± 0.26
18 ± 0.03
22 ± 0.06
26 ± 0.25
24 ± 0.66
VA
27 ± 0.58
20 ± 0.26
Interpretative criteria (CLSI) Zone inhibition diameter (mm)
AM
E
P
TE
VA
S- > 17
I-Nil
R- < 15
S- > 23
I-14-22
R- < 13
S- > 15
I-Nil
R- < 14
S- > 19
I-15-18
R- < 14
S- > 17
I-15-16
R- < 14
Note: AM: ampicillin, E: erythromycin, P: penicillin G, TE: tetracycline, VA:
vancomycin. S: Sensitive; I: Intermediate; R: Resistant. The mean of three values
of zone of growth inhibition of each antibiotic are presented along with ± SD.
4
D. Vadassery Haridas et al.
Aquaculture 548 (2022) 737597
Viable cell count (log10 CFU/mL
9.00
8.00
7.00
6.00
5.00
4.00
L.plantarum QQ8
3.00
L.casei QQ10
2.00
1.00
0.00
0%
0.30%
0.60%
0.90%
Bile concentration
Viable cell count (log10 CFU/mL
Fig. 1. Bile tolerance of QQ isolates- Effect of bile salt on the growth of two QQ isolates (L. plantarum QQ8 and L. casei QQ10) at 30 0C. To check bile-salt tolerance,
the survival of QQ isolates in suitable growth medium with different concentration of bile salt for 18 h was determined by the viable cell counts on agar plates. Values
are mean±SD of three different observations.
10.00
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
L.plantarum QQ8
L.casei QQ10
pH1
pH2
pH3
pH4
pH5
pH6
pH7
Different pH
Fig. 2. pH tolerance of QQ isolates- Effect of pH on the growth of L. plantarum QQ8 and L. casei QQ10 at 30 0C. To check pH resistance, the survival of QQ isolates
in suitable growth medium with different pH for 18 h was determined by the viable cell counts on agar plates. Values are mean±SD of three different observations.
3.4. Quantification of AHL degradation by microtiter plate assay
3.6. Experimental challenge with A. hydrophila
The AHL degradation by four QQ isolates was quantified by micro­
titer plate assay. All isolates showed high level of inhibition of violacein
production (AHL degradation) (Fig. 6). The highest rate of.
inhibition was noticed in the mixture of four QQ isolates (90%)
followed by B. thuringiensis QQ17 (84%) (Fig. 6). ANOVA showed that
there was significant difference (p ≤ 0.01) in the AHL degradation rate
between mixture of QQ isolates and individual isolate. Post Hoc analysis
using Duncan’s Multiple Range Test grouped the QQ isolates into three
homogenous groups viz.; (1) L. plantarum QQ8, L. casei QQ10 and
E. faecium QQ12 (had inhibition of 81%, 83% and 81% respectively) (2)
B. thuringiensis QQ17 (had 84% inhibition) and (3) mixture of four QQ
isolates (had 90% inhibition) (Fig. 6).
The results obtained are given in Table 1. In control groups,
following challenge with A. hydrophila, all fish showed severe skin le­
sions and 90–100% mortality was observed in two weeks. The admin­
istration of probiotic diet in all combinations afforded effective
protection in goldfish against experimental A. hydrophila infection.
Among the 28 groups of goldfish fed with 28 different probiotic com­
binations each, 26 showed 50% -90% of survival at the end of two
weeks. The low survival rate (below 50%) was noticed only in two
groups of goldfish (First group fed with 107 CFU/g feed of L.plantarum
QQ8 and 103 CFU/g feed of E. faecium QQ12 and second group fed with
107 CFU/g feed of L. plantarum QQ8 and 103 CFU/g feed of L. casei
QQ10). A. hydrophila was isolated from haemorrhagic lesions of both
dead and survived fish.
The fitted cubic model was found to be significant (p < 0.05) to
explain the variability in the survival rate as a function of input vari­
ables. The R2 and RMSE values of fitted model were 0.92 and of 5.57,
respectively. The linear, quadratic and cubic regression coefficients of
fitted model were significant at 5% of level of significance. The esti­
mated values of regression coefficients of fitted model are given in
Table 3.
The response surface plots of survival rate is given in Fig. 7A, B, C
3.5. Safety of the QQ8 and QQ10
The administration of L. plantarum QQ8 and L. casei QQ10 even at the
concentration of 1 × 1010 cells per fish did not result in any unfavorable
effect on fish activity. All fish were clinically healthy and behaved no
different from the control group, suggesting that the QQ isolates are not
pathogenic to fish.
5
D. Vadassery Haridas et al.
Aquaculture 548 (2022) 737597
1.00
Absorbance @ 600nm
0.90
0.80
0.70
0.60
0.50
L.plantarum QQ8
0.40
L.casei QQ10
0.30
0.20
0.10
0.00
0%
0.20%
0.50%
Phenol %
Fig. 3. Phenol tolerance of QQ isolates- Effect of phenol on the growth of two QQ isolates (L. plantarum QQ8 and L. casei QQ10) at 30 0C. To check phenol
resistance, the growth of QQ isolates in suitable growth medium for 18 h was determined by measuring OD at 600 nm after adjusting the culture media to the specific
phenol concentration. Values are mean±SD of three different observations.
Autoaggregation percentage
90.00
80.00
70.00
60.00
50.00
L.plantarum QQ8
40.00
L.casei QQ10
30.00
20.00
10.00
0.00
1h
2h
3h
4h
5h
Time
Fig. 4. Auto-aggregation rate of QQ isolates- In the QQ isolates aggregation percentage increased every 1h and highest rate was observed at 5 h.
and D.The optimum dosage of probiotic combination was obtained by
the desirability score (Table 3). The efficiency of this multispecies pro­
biotic combination composed of 103 CFU/g feed of L. plantarum QQ8,
102 CFU/g feed of L. casei QQ10, 102 CFU/g feed of E. faecium QQ12 and
103 CFU/g feed of B. thuringiensis QQ17 was validated by the feeding and
challenge experiment. The high rate of survival (91.7%) in goldfish
against A. hydrophila infection (Table 4) confirmed the potency of newly
derived probiotic mixture. The desirability graph is given in Fig. 8.
pathogenic bacteria in aquaculture (Defoirdt et al., 2007). The results of
the present study showed that, in addition to possessing excellent
quorum quenching property, L. plantarum QQ8 and L. casei QQ10 iso­
lated from tilapia gut, have very good probiotic properties such as bile
salts, acid and phenol resistance, auto-aggregation, co-aggregation and
antibiotic sensitivity. Acid and bile tolerance are two inevitable prop­
erties that give a probiotic the potential to remain alive in the upper
gastrointestinal tract (Erkkila and Petaja, 2000; Hyronimus et al., 2000).
In the present study, the quorum quenching isolates L. plantarum QQ8
and L. casei QQ10 tested for bile salt tolerance exhibited survival even in
0.9% bile salt at 18 h of incubation and there were no significant dif­
ferences in log10 CFU/ ml between control and different concentrations
of bile salt treatment suggesting that they have the capacity to withstand
the conditions in fish gut. Many reports are found to describe the bile salt
tolerance of L. plantarum (Erwann et al., 2011; Hassan et al., 2012) and
L. casei (Hassan et al., 2012; Soliman et al., 2015). When a bacterium
prefers a pH above 5.5, but able to grow at pH 5.5 or below, it can be
4. Discussion
The present study focused on fish gut bacteria L. plantarum QQ8 and
L. casei QQ10 that exhibited both probiotic and quorum quenching
ability. To the best of our knowledge, there are hardly any reports
demonstrating the quorum quenching ability of probiotic L. plantarum
and L. casei. The implementation of quorum quenching property to
choose a new probiotic gives an alternative to antibiotics to control
6
D. Vadassery Haridas et al.
Aquaculture 548 (2022) 737597
5h
Coaggregation percentage
45.00
40.00
35.00
30.00
25.00
20.00
5h
15.00
10.00
5.00
0.00
L.plantarum QQ8
L.casei QQ10
QQ isolates
Violacien inhibition % (AHL degradation)
Fig. 5. Co-aggregation rate of QQ isolates- QQ bacteria exhibited very good co-aggregation ability after 5 h of incubation with A. hydrophila.
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
-10%
Violacien inhibition %
QQ Isolates
Fig. 6. Quantification of AHL degradation by microtiter plate method. Graph represents the percentage of inhibition of violacein production in C.violaceum by
different QQ isolates (L. plantarum QQ8, L. casei QQ10, E. faecium QQ12, B. thuringiensis QQ17 and mixture of isolates). Vertical bars represent the AHL degradation.
Values are mean±SD of triplicate observations. The percentage of inhibition of violacein production between individual QQ isolate and mixture of isolates was
significantly different. (p≤0.01).
classified as acid tolerant. Fish gastrointestinal pH shows great variation
among species with a range of 1.47 to 5.12 and the lowest value
observed was 1.18 (Welliton et al., 2017). However, such extreme low
pH is transient. The pH value raises to 3 and above in the presence of
food (Erkkila and Petaja, 2000). Soliman et al. (2015) showed that there
was no reduction in viability when L. casei and L. plantarum were
exposed to pH 3.0 over 3 h, indicating a high level of acid tolerance in L.
casei and L. plantarum. In the present study, we found that L. plantarum
QQ8 and L. casei QQ10 grew at pH 3 or above. This result indicates that
the QQ isolates given as a probiotic diet will be able to survive the harsh
conditions of the gut environment and colonize the intestinal tract,
thereby will be capable of imparting their benefits. In this study the QQ
isolates could also persist well at 0.5% of phenol in MRS media. Phenol
may be synthesized in the intestine by bacterial deamination of various
aromatic amino acids obtained from dietary or endogenously derived
protein (Suskovic et al., 1997). Studies on different animal models
reveal that phenol has a bacteriostatic effect against gut bacteria (Hoier,
1992). Since probiotics should withstand the harsh gut environment,
tolerance to phenol is considered as a mandatory probiotic property
(Perez-Miranda et al., 2007).
Auto-aggregation and co-aggregation properties are considered as
important characteristics of probiotic bacteria. Assessment of autoaggregation and potential to co-aggregate with harmful intestinal
pathogens can be used for initial evaluation and selection of the best
probiotic strain. Bao et al. (2010) stated that auto-aggregation property
of probiotic bacteria is responsible for the bacterial adhesion on to the
intestinal cell wall; an essential feature for colonization of probiotic
strains in the gastrointestinal tract. Jelena and Natasa (2015) tested the
auto-aggregation property of 12 lactic acid bacteria and among them
E. faecium and L. plantarum strains had the highest degree of autoaggregation (44% and 50%, respectively). In this study, the L. planta­
rum QQ8 (77.73 ± 0.13%) and L. casei QQ10 (66.73 ± 0.04%) exhibited
7
D. Vadassery Haridas et al.
Aquaculture 548 (2022) 737597
Table 3
Estimated regression coefficients of fitted model.
Isolates
QQ8
QQ10
QQ12
QQ17
QQ8 * QQ10
QQ8 * QQ12
QQ8 * QQ12
QQ8 * QQ17
QQ10 * QQ12
QQ10 * QQ17
QQ12 * QQ17
QQ8 * QQ10 * QQ12
QQ8 * QQ10 * QQ17
QQ8 * QQ12 * QQ17
QQ10 * QQ12 * QQ17
Regression coefficients
Estimated values
β1
β2
β3
β4
β12
β13
β13
β14
β23
β24
β34
β123
β124
β134
β234
+7.83450
+6.78250
+8.48882
+7.71805
-1.23120
-1.50049
-1.50049
-0.85457
-0.72078
-0.47599
-1.12845
+1.39807
+1.15189
+1.36477
+0.92592
Table 4
Survival percentages of Carassius auratus with optimized
probiotic diet.
Probiotics
Survival %
Control
Optimized probiotic
16.7 ± 5.8
91.7 ± 0
Note: Values are mean ± SD of triplicate observations.
high degree of auto-aggregation. Co-aggregation ability, also called
“barrier effect” of probiotics, might become an obstacle that prevents
colonization of pathogenic bacteria in the gastrointestinal tract (Garcia
et al., 2014). In order to measure interbacterial adherence, Blagoeva
et al. (2014) examined the co-aggregation of 34 LAB strains with two
pathogenic strains (Salmonella sp. NBIMCC 1425 and Listeria mono­
cytogenes NBIMCC 8669). Among them L. plantarum and E. faecium
showed highest rate of co-aggregation (27.31% and 24.03% respec­
tively) with Salmonella sp. NBIMCC 1425. In the present study,
L. plantarum QQ8 (39.61 ± 0.23%) and L. casei QQ10 (29.4 ± 0.18%)
demonstrated good co-aggregation ability with A. hydrophila. This
Fig. 8. Desirability score plot as a function of L. plantarum QQ8, E. faecium
QQ12 and L. casei QQ10 and keeping B. thuringiensis QQ17 constant.
Fig. 7. A. Response surface plot of survival rate as a function of L. plantarum QQ8, E. faecium QQ12 and B. thuringiensis QQ17 keeping L. casei QQ10 constant.
B. Response surface plot of survival rate as a function of L. plantarum QQ8, L. casei QQ10 and B. thuringiensis QQ17 keeping E. faecium QQ12 constant.
C Response surface plot of survival rate as a function of L. casei QQ10, E. faecium QQ12 and B. thuringiensis QQ17 keeping L. plantarum QQ8 constant.
D Response surface plot of survival rate as a function of L. plantarum QQ8, L. casei QQ10 and E. faecium QQ12 keeping B. thuringiensis QQ17 constant.
8
D. Vadassery Haridas et al.
Aquaculture 548 (2022) 737597
ability of QQ isolates might be a reason for the prevention of coloniza­
tion of A. hydrophila in the goldfish gastrointestinal tract.
The antibiotic sensitivity of bacteria is considered as one of the
important strategy to develop safe probiotic products for aquaculture
applications. Antibiotic resistance in probiotic bacteria may result in
active transfer of antibiotic resistant genes from probiotics to other in­
testinal microflora and finally to opportunistic pathogens that reside in
the same harsh environment. The bacteria which are used as probiotics
should be free from acquired antibiotic resistance genes to prevent
lateral spread of resistance (Peres et al., 2014). In the present study,
L. plantarum QQ8 and L. casei QQ10 exhibited susceptibility to all five
antibiotics tested. This result supports the possibility of these isolates to
be developed as probiotic.
In this study, the AHL degradation ability of the quorum quenching
isolates L. plantarum QQ8, L. casei QQ10, E. faecium QQ12 and
B. thuringiensis QQ17 was quantified by microtiter plate assay and per­
centage inhibition of violacein production by each isolate individually
and in combination was determined. The increased AHL degrading ca­
pacity expressed by the mixture of four QQ isolates, that was capable of
inhibiting violacein production in CV026 by 90%, showed the impor­
tance of using quorum quenching isolates in combination rather than
using as single isolate for protection against pathogenic bacteria. It is
suggested that the enhanced AHL degrading effect of the four QQ iso­
lates in combination might be due to the synergistic effect of these
different strains which resulted in the production of more lactonase
enzyme.
In aquaculture, nearly all studies on the potency of probiotic bacteria
concentrated on single strains. There are far fewer studies that focus on
the efficacy of multi-species probiotics. In the present study, the com­
bined effects of L. plantarum QQ8, L. casei QQ10, E. faecium QQ12 and
B. thuringiensis QQ17 in reducing A. hydrophila infection in goldfish
C. auratus was investigated by using a randomized experimental model
of response surface methodology. Many scientific studies on animal
health state that probiotic mixtures have favourable effects against
pathogenic attack and the mixture of different probiotic bacteria in a
product can confer significant protection against several intestinal
pathogens compared to single strain products (Chang et al., 2017; Seyed
et al., 2019; Wang et al., 2019). In the present study, multi-species diet
with all the 28 tested combination for 30 days protected the fish when
challenged with A. hydrophila and showed 40% -90% of survival at the
end of two weeks. The best probiotic combination was optimized by
response surface methodology. The validation of the optimum probiotic
combination derived from the statistical analysis showed that the pro­
biotic combination of 103 CFU/g feed of L. plantarum QQ8, 102 CFU/g
feed of L. casei QQ10, 102 CFU/g feed of E. faecium QQ12 and 103 CFU/g
feed of B. thuringiensis QQ17 provided maximum protection in goldfish
against A. hydrophila infection with 91.7% survival after the bacterial
challenge. This result was comparable to findings by Wang et al. (2019),
that the multi strain probiotic containing Lactobacillus pentosus BD6,
Lactobacillus fermentum LW2, B. subtilis E20, and Saccharomyces cer­
evisiae P13 at a dose of 10 8 CFU/Kg feed for 56 days could improve
growth and health status in white shrimp, Litopenaeus vannamei better
than when single probiotics were used. They noticed higher survival in
shrimp fed with probiotic mixture after challenge with Vibrio alginoly­
ticus and suggested that potency of probiotic mixture was due to syn­
ergistic interactions between bacterial strains.
It is evident that, in a probiotic mixture, the component strains work
in a synergistic way where they mutually enhance the activity of each
strain and induce substantial probiotic effect. It is very crucial that, in
multi-strain probiotics the component strains do not act as antagonists.
The interspecies inhibition exhibited occasionally by probiotic mixture
may potentially reduce their beneficial effects (Chapman et al., 2012).
Better protection afforded by the four strain probiotic mixture evidenced
by the higher survival rate in gold fish after challenging with
A. hydrophila clearly suggest that component strains of the probiotic
mixture used in the present study do not have any inter species
antagonism.
5. Conclusion
Production of AHL degrading enzyme by the QQ isolates might have
reduced the pathogenicity of A. hydrophila, while, their probiotic prop­
erties might have simultaneously helped them to out-compete
A. hydrophila for nutrients and space and exclude the pathogenic bac­
teria through antagonistic activity. Thus, the results of the in vitro and in
vivo studies lead us to conclude that the higher survival rate of goldfish
fed with probiotic mixture is due to the combined effect of quorum
quenching ability of the strains together with their probiotic activity.
Statement of ethical approval
The present research involving fish was carried out in strict accor­
dance with the guidelines of the Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA). The protocol was
reviewed and approved by the institutional animal ethics committee of
Kerala University of Fisheries and Ocean Studies, India. All dissection
was performed under tricaine methanesulfonate (Sigma) (100 mg L-1)
anesthesia and all efforts were made to minimize suffering.
Declaration of Competing Interest
None.
Acknowledgments
The authors thank the authorities of the Kerala University of Fish­
eries and Ocean Studies (KUFOS), Kochi, Kerala for the facilities
extended for carrying out this work. This work was supported by the
Plan fund at the Centre of Excellence for Sustainable Aquaculture and
Aquatic Animal Health Management, KUFOS.
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