animals

Article
Limited Probiotic Effect of Enterococcus gallinarum L1,
Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3 to
Protect Rainbow Trout against Saprolegniosis
Juan-Miguel Fregeneda-Grandes 1, * , Concepción González-Palacios 1 , Tania Pérez-Sánchez 2 ,
Daniel Padilla 3 , Fernando Real 3 and José-Miguel Aller-Gancedo 1

1 Departamento de Sanidad Animal, Campus de Vegazana s/n, Universidad de León, 24071 León, Spain
2 Departamento Agricultura y Alimentación, Universidad de La Rioja, Avenida Madre de Dios 53,
26006 Logroño, Spain
3 Instituto Universitario de Sanidad Animal y Seguridad Alimentaria (IUSA), Universidad de Las Palmas de
Gran Canaria, Carretera de Trasmontaña s/n, 35416 Araucas, Spain
* Correspondence: [email protected]

Simple Summary: Probiotics have been considered as alternatives to the antibiotics currently used
to control diseases caused by different microorganisms, which show high prevalence and losses
in aquaculture. In this study, the possible utility of three probiotics (Enterococcus gallinarum L1,
Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3—effective against vibriosis or lactococosis
in sea bass or rainbow trout) was investigated for the biocontrol of saprolegniosis in rainbow trout.
For this purpose, both in vitro inhibition studies and competition for binding sites against Saprolegnia
parasitica and in vivo tests with experimentally infected rainbow trout were carried out. Although the
three probiotics showed inhibitory capacity and reduced the adhesion activity of S. parasitica cysts to
cutaneous mucus in vitro, none of the three bacteria showed in vivo protection through either water
or feed. The obtained results show the importance of selecting the most appropriate probiotic and its
mechanism of action depending on the species of fish and the disease to be prevented.

Abstract: Previous studies have demonstrated that the strains Enterococcus gallinarum L1, Vagococcus
Citation: Fregeneda-Grandes, J.-M.;
fluvialis L21 and Lactobacillus plantarum CLFP3 are probiotics against vibriosis or lactococosis in sea
González-Palacios, C.; Pérez-Sánchez,
bass or rainbow trout. In this study, the utility of these bacterial strains in the control of saprolegniosis
T.; Padilla, D.; Real, F.; Aller-Gancedo,
J.-M. Limited Probiotic Effect of
was evaluated. For this purpose, both in vitro inhibition studies and competition for binding sites
Enterococcus gallinarum L1, Vagococcus against Saprolegnia parasitica and in vivo tests with experimentally infected rainbow trout were carried
fluvialis L21 and Lactobacillus out. In the in vitro tests, the three isolates showed inhibitory activity upon mycelium growth and cyst
plantarum CLFP3 to Protect Rainbow germination and reduced the adhesion of cysts to cutaneous mucus; however, this effect depended
Trout against Saprolegniosis. Animals on the number of bacteria used and the incubation time. In the in vivo test, the bacteria were
2023, 13, 954. https://doi.org/ administered orally at 108 CFU g−1 in the feed or at 106 CFU ml−1 in the tank water for 14 days.
10.3390/ani13050954 None of the three bacteria showed protection against S. parasitica infection either through water or
Received: 2 February 2023 feed, and the cumulative mortality reached 100% within 14 days post infection. The obtained results
Revised: 2 March 2023 show that the use of an effective probiotic against a certain disease in a host may not be effective
Accepted: 5 March 2023 against another pathogen or in another host and that the results obtained in vitro may not always
Published: 6 March 2023 predict the effects when used in vivo.

Keywords: biocontrol; probiotics; saprolegniosis; inhibition assays; Oncorhynchus mykiss

Copyright: © 2023 by the authors.

Licensee MDPI, Basel, Switzerland.
This article is an open access article
1. Introduction
distributed under the terms and
conditions of the Creative Commons Saprolegniosis is a fungal disease, the main pathogenic agent of which is Saprolegnia
Attribution (CC BY) license (https:// parasitica, that can cause heavy economic losses in inland fish farms due to depletion of
creativecommons.org/licenses/by/ fish stock and eggs [1]. This disease affects freshwater fish at any stage of development
4.0/). and is characterized by skin lesions such as patches with a cotton wool-like appearance.

Animals 2023, 13, 954. https://doi.org/10.3390/ani13050954 https://www.mdpi.com/journal/animals

Animals 2023, 13, 954 2 of 15

Current prevention and treatment measures for saprolegniosis rely on the use of chemicals
products that may cause damage to the environment [2,3] and humans [4]. In addition,
none of the available compounds offers enough protection after the rearing period [5].
Recently, metal-based nanoparticles have attracted attention as a potential material for
prevention and control of saprolegniosis [6,7]. Among the biological control alternatives to
chemotherapy is the use of plant extracts [8–12] or probiotics [13,14].
In this sense, a number of in vitro studies have evidenced that some bacterial strains
can inhibit Saprolegnia spp., including Pseudomonas fluorescens [15–19], Alteromonas sp.,
Pseudomonas alcaligenes, Pseudomonas saccharophila, Aeromonas caviae and Aeromonas
eucrenophila [20], Serratia marcescens [21], Bacillus subtilis [22], Aeromonas media [23], Aeromonas
sobria, Pantoea agglomerans, Serratia fonticola, Xhantomonas reflexus and Yersenia kristensenii [17,18],
Lactobacillus plantarum [24] and Pseudomonas aeruginosa [25]. However, their action in vivo has
only been studied in eel Anguilla australis [13], silver perch Bidyanus bidyanus [14] and eggs of
rainbow trout Oncorhynchus mykiss [26] or eggs of gourami Osphonemus gouramy [27].
In previous studies by our research group, various bacterial isolates with an in vitro
ability to inhibit the growth of S. parasitica were obtained [17]. Their harmless nature with
respect to rainbow trout (Oncorhynchus mykiss) and their ability to adhere the cutaneous
mucus and reduce the adhesion of zoospores and cysts of S. parasitica were also demon-
strated [18]. It was also found that of the fifteen investigated isolates, under experimental
conditions, two isolates of Pseudomonas fluorescens reduced infection by S. parasitica in
rainbow trout when these bacteria were added to the water in the tanks [28], and the mode
of action of these bacteria was likely associated with the production of siderophores [29].
Enterococcus gallinarum L1 (obtained from Dicentrarchus labrax gut content) and
Vagococcus fluvialis L21 (obtained from Solea solea gut content) demonstrated high lev-
els of protection against Vibrio anguillarum in European sea bass (Dicentrarchus labrax) after
an experimental challenge when these bacteria were added to feed for 20 days [30,31]. In
the same way, Lactobacillus plantarum CLFP-3 (obtained from O. mykiss cutaneous mucus)
added to the diet of rainbow trout for 36 days was demonstrated to improve protection
against Lactococcus garvieae [32]. In the present study, the in vitro inhibitory activity of these
probiotic bacteria against S. parasitica was determined, and in vivo tests with experimen-
tally infected rainbow trout were carried out in order to investigate their potential use for
the biocontrol of saprolegniosis.

2. Materials and Methods

2.1. Collection of Isolates Used in the Study
Three probiotic bacteria (Enterococcus gallinarum L1, Vagococcus fluvialis L21 and Lactobacillus
plantarum CLFP3) previously obtained by our research group that were stored at −80 ◦ C in our
laboratory facilities were used in this study (Table 1). The first two have shown their efficacy
against infection by Vibrio aguillarum in sea bass (Dicentrarchus labrax), and L. plantarum reduced
mortality in rainbow trout (O. mykiss) infected with Lactococcus garvieae.

Table 1. Probiotic bacterial strains tested in the present study for biocontrol of saprolegniosis in
rainbow trout (Oncorhynchus mykiss).

Bacterial Isolate Origin Probiotic Activity Against Reference

Enterococcus gallinarum L1 Gut content of sea bass (Dicentrarchus labrax) Vibrio angillarum in D. labrax [31]
Vagococcus fluvialis L21 Gut content of sole (Solea solea) Vibrio angillarum in D. labrax [30]
Lactobacillus plantarum subsp. plantarum CLFP3 Cutaneous mucus of rainbow trout (O. mykiss) Lactococcus garvieae in O. mykiss [32]

After thawing, the bacterial isolates E gallinarum L1 and V. fluvialis L21 were cultured
overnight at 20 ◦ C in 3 mL of brain–heart infusion broth (BHIB, Pronadisa, Condalab,
Madrid, Spain), while L. plantarum CLFP3 was cultured in Man, Rogosa and Sharpe broth
(MRS, Pronadisa). An aliquot of 500 µL of the culture was inoculated into 25 mL of BHIB
or MRS broth and incubated at 20 ◦ C and 200 rpm on a rotary orbital shaker (Innova® 44,
New Brunswick Scientific, Edison, NJ, USA) until the middle of the exponential growth
phase (based on previously established growth curves). Bacteria were pelleted and washed
Animals 2023, 13, 954 3 of 15

twice by centrifuging at 1000× g for 15 min and resuspended in sterile saline solution. The
number of bacteria was adjusted to the desired concentration according to the experiment
in which they were used (see below) by counting in a hemocytometer chamber (Improved
Neubauer Type, Albert Sass, Germany).
The strain Saprolegnia parasitica TRU12 isolated from a wild brown trout (Salmo trutta)
with saprolegniosis [33] and maintained refrigerated in our laboratory was used. It was
cultured for 3 d at 20 ◦ C on glucose peptone (GP) agar; then, autoclaved half hemp seeds
of Cannabis sativa were placed on the edges of the colony, followed by an additional 24 h of
incubation at 20 ◦ C. To obtain zoospores, the half hemp seeds colonized by hyphae were
placed in Petri dishes with filtered autoclaved river water. After 36–48 h at 20 ◦ C, the water
was filtered through Whatman 541 cellulose filter paper. The number of zoospores was
then estimated using a Hawksley Cristalite BS 748 counting chamber.

2.2. In Vitro Inhibition Assays of the Probiotic Bacteria against S. parasitica

To determine the in vitro inhibition of the probiotic bacteria strains against S. parasitica,
different assays were performed. All these experiments were conducted following the
procedures described in a previous study [17].

2.2.1. Inhibition of Hyphal Growth on Solid Media (Plate Assay)

This test was performed in with BHI agar (Cultimed, Panreac Química SLU, Barcelona,
Spain), whereby each bacterium was streaked twice. This assay was performed in triplicate
with incubations at 3, 5 or 7 d at 20 ◦ C. Then, a 3 mm diameter block of agar with young
hyphal tips of S. parasitica was placed between the 2 bacterial streaks and incubated for 3 d
at 20◦ C, measuring the diameter of the S. parasitica colony. A negative control plate was
used without bacterial streaks under the same conditions.

2.2.2. Inhibition of Hyphal Growth from Colonized Hemp Seeds and Cyst Germination in
Liquid Media
Bacterial dilutions, hemp seeds colonized by Saprolegnia and zoospore suspensions
were prepared following the methods described above. The bacterial concentration was
adjusted to 2 × 105 cells mL−1 , and, taking this as the first dilution, four 10-fold dilutions
of bacterial suspension were prepared. The concentration of zoospores was adjusted to
4 × 104 zoospores mL−1 . For the inhibition of hyphal growth, duplicate wells of a 24-well
tissue culture plate (Falcon, Corning, Glendale, AZ, USA) filled with 1 mL of each bacterial
dilution, 1 mL of BHIB and a half hemp seed colonized by S. parasitica were dispensed into
each well.
For the inhibition of zoospores/cyst germination, the same procedure was followed, with
the only difference being that 0.5 mL of the zoospore suspension (4 × 104 zoospores mL−1 )
was added in place of a colonized hemp seed, and 0.5 mL of each bacterial dilution was
used instead of 1 mL.
In both assays, plates were incubated for 3 d at 20 ◦ C, and bacteria and S. parasitica
zoospore negative controls were included on each plate. The presence or absence of
macroscopic or microscopic hyphal growth and germination of cysts were observed by a
Nikon Diaphot inverted microscope.

2.2.3. Assay to Evaluate Fungicidal Effects

The initial step was the same as that for the hemp seed test, but the plates were placed
in an incubator at 20 ◦ C for 1, 2 or 3 d. Following each incubation period, the hemp seeds
from the wells where the growth of S. parasitica was inhibited were removed, washed
3 times with sterile distilled water and transferred to a Petri dish containing 20 mL of
filtered and autoclaved river water with streptomycin (2 × 102 mg L−1 ) and penicillin
(2 × 105 UI L−1 ). The plates were incubated for 20 d at 20 ◦ C. To avoid false-positive
results due to inhibitory effects of viable bacteria in the mycelium, only the results with no
bacterial growth in water samples collected after 24 h were accepted. A negative control
Animals 2023, 13, 954 4 of 15

was achieved by incubating a hemp seed colonized by S. parasitica in water both with and
without antibiotics. The bacteria were considered to have a fungicidal effect if no mycelial
growth was observed after 20 d.

2.2.4. Inhibition Assays with Bacterial Culture Supernatants

The supernatants used to perform this assay were prepared according to Lategan et al. [34]
using BHIB for L1 and L21 or MRS for CLFP3 strains. This supernatant and three 2-fold
serial dilutions were tested to analyze the ability of the supernatants to inhibit hyphal
growth (hemp-seed test) and to evaluate the capacity of the supernatants to inhibit cyst
germination (cyst test). In the hemp-seed test, 2 mL of each supernatant dilution and a half
hemp seed colonized by S. parasitica were dispensed in duplicate into each well of a 24-well
tissue culture plate. In the cyst test, 1 mL of each supernatant dilution and 1 mL of the
zoospore suspension (4 × 104 zoospores ml−1 ) were added to each well. Incubation times
and observation procedures were the same as those described in Section 2.2.2. Negative
controls with only culture medium were included in both assays.

2.3. Adhesion to Skin Mucus of the Probiotic Bacteria and Inhibition of S. parasitica Cyst Adhesion
The adhesion capacity of the probiotic strains L1, L21 and CLFP3 to cutaneous mucus
of trout and its potential to hinder the adhesion of S. parasitica cysts under conditions of
exclusion, competition and displacement were investigated using the methods described
by Carbajal-González et al. [18].
Cutaneous mucus was obtained from four male brown trout (Salmo trutta) with an av-
erage body weight of 196.7 ± 39.5 g from a hatchery belong to the Regional Government of
Castile and Leon by scraping the surface with a plastic spatula. The mucus was centrifuged
twice at 12,000× g for 5 min at 4 ◦ C to eliminate particulate and cellular debris. The protein
concentration was adjusted to 0.05 mg ml−1 in PBS using Bradford reagent (Sigma-Aldrich,
Merck Life Science, Madrid, Spain). The resulting mucus suspension was finally sterilized
by UV light exposure for 30 min and stored in aliquots at −20 ◦ C until use.
Bacteria and cysts of S. parasitica were stained with Syto 9® green fluorescent nucleic
acid stain (Invitrogen, Fisher Scientific, Madrid, Spain) at a concentration of 1 µL per
109 bacteria in 1 mL and 1 µL per 105 cysts in 1 mL.

2.3.1. Adhesion to Skin Mucus of the Probiotic Bacteria

This assay was performed in 96-well black polystyrene plates (Costar, ImmunoChem-
istry Technologies, Davis, CA, USA) coated with cutaneous mucus. In the adhesion assay
with the probiotic strains, wells without coating or coated with BSA (0.05 mg ml−1 , Sigma-
Aldrich) or mucin from swine stomach type II (MSS; 0.05 mg ml−1 , Sigma-Aldrich) were
used as controls for non-specific adhesion. To coat the plates, 25 µL of mucus, BSA or mucin
and 75 µL of coating buffer (16.8 g sodium hydrogen carbonate, 21.2 g sodium carbonate
per liter, pH 9.6) were added to each well and left overnight at 4 ◦ C. Subsequently, the wells
were washed with PBS-Tween 20 (0.1%), the fluorescently labelled bacterial solution (25 µL
of 109 bacterial cells ml−1 ) was added and plates were centrifuged at 163× g for 12 s to
promote the adherence of bacteria to the mucus. Bacterial fluorescence was measured with
a spectrophotometer (Synergy HT Multi-Detection Microplate Reader, Bio-Tek® , Winooski,
VT, USA) at 485 nm excitation and 535 nm emission after 30 min of incubation in darkness
and at room temperature (time 0). Non-attached bacteria were removed by washing with
saline solution (50 µL/well), and plates were placed upside down on absorbent paper and
centrifuged to remove any remaining liquid. Then, 50 µL of saline solution was added per
well, and a new measurement was performed. Adhesion was expressed as the percentage
of fluorescence recovered in the second measurement relative to the fluorescence at time 0.

2.3.2. Inhibition of S. parasitica Cyst Adhesion

Seven serial 10-fold dilutions were performed from non-stained bacteria (2 × 109 cells mL−1)
in saline solution. The cysts were stained using the method mentioned previously using
Animals 2023, 13, 954 5 of 15

a concentration of 105 cysts mL−1 . In the exclusion test, each dilution of the bacterial
isolates was added to the wells, and after being incubated and washed, the labelled cysts
were added. In the competition test, both bacterial dilution and stained cysts were added
simultaneously, and in the displacement test, the labelled cyst suspension was first added,
incubated and washed; then, each dilution of the bacterial suspension was added. Plates
were washed with saline solution after 60 min of incubation at room temperature for each
condition; then, the fluorescence was recorded. Wells with saline solution instead of bacte-
rial cells were used as controls in this assay. The percentage of cysts bound to these control
wells was considered the reference value (100%), and the percentage of cyst adhesion in the
presence of bacteria was compared to this reference value.

2.3.3. Statistical Analysis

In the adhesion assays, statistical analyses were carried out using SPSS for Windows
version 26 (IBM SPSS Statistic, Armonk, NY, USA) by the Student’s t-test (p ≤ 0.05). Data
were shown as the mean ± standard deviation of three separate trials.

2.4. Pathogenicity for Rainbow Trout

The probiotic bacterium CLFP3 (L. plantarum), which has previously been used suc-
cessfully to prevent lactococcosis in trout [32], was obtained from rainbow trout. However,
the L1 (E. gallinarum) and L21 (V. fluvialis) bacteria were obtained from marine fish, and
although they were previously reported to be non-pathogenic for sea bass [30,31], their
potential pathogenicity for rainbow trout is unknown. Therefore, prior to being used in
in vivo tests, experimental inoculations with L1 and L21 isolates were conducted to verify
their lack of pathogenicity for rainbow trout.
Rainbow trout (O. mykiss) from a commercial fish farm with an average body weight
of 37.24 ± 7.56 g were acclimatized for 10 days in a 120 L tank and observed daily to
ensure that they did not show clinical signs of disease. Trials were conducted using groups
of 20 fish kept in 40 L tanks filled with chlorine-free well water with a renewal rate of
1.5 L per h at 12 ◦ C with constant aeration and a photoperiod of 12/12 h. Effluent water
was disinfected with ozone. The experimental protocol was approved by the Subcommit-
tee for Experimentation and Animal Welfare of the University of Leon, Spain (protocol
number ULE_04_2015).
The bacterial isolates were grown in BHIB as described in Section 2.1 to obtain a
suspension of 107 cells mL−1 . Fish were anaesthetized with tricaine methanesulfonate
(MS-222, 50 mg mL−1 ) and injected with 0.1 mL intraperitoneally (ip) and intramuscularly
(im) in two separate groups of 20 trout each. Control groups of 20 trout were inoculated
with 0.1 mL of saline solution at the same time points. Fish were observed for 10 days and
euthanized by overdosing them with MS-222 (100 mg mL−1 ) at the end of the observa-
tion period. They were subsequently examined for evidence of disease (i.e., gross lesions).
Additionally, loopfuls of material from the kidney, liver and spleen were spread over plates
of BHI agar with incubation at 20 ◦ C for 3 days to determine the presence or absence of the
inoculated bacterial isolates in the fish. The recovered bacterial isolates were examined by
means of Gram-stained smears and basic biochemical tests such as catalase and oxidase tests.
Temperature and other physicochemical parameters of water were measured daily,
and no significant differences were observed between the different groups (temperature,
12.47 ± 0.24 ◦ C; pH 7.92 ± 0.06; dissolved oxygen, 9.72 ± 0.16 mg L−1 ; nitrites (NO2 ),
0.034 ± 0.005 mg L−1 ; and non-ionized ammonia (NH3 ), 0.059 ± 0.005 mg L−1 ).

2.5. Biocontrol of Saprolegniosis by Adding Bacteria to Water or Feed

2.5.1. Biocontrol by Adding Bacteria to Water
For each of the tested bacteria, 60 rainbow trout with an average body weight of
28.79 ± 10.01 g in three 40 L tanks were used: 20 rainbow trout for treatment with the
probiotic and 2 as controls for infection and ami momi treatment, respectively. Fish were
infected with S. parasitica TRU12 following the method described previously by Fregeneda-
Animals 2023, 13, 954 6 of 15

Grandes et al. [33]. Briefly, the fish were slightly scarified on the skin using a variation
of ami momi treatment [35]. Groups of 10 fish were shaken for 2 min in a cylindrical
stainless-steel colander with a mesh of 7 mm before adding a suspension of S. parasitica
zoospores to the water to obtain a final concentration of 3 × 102 spores mL−1 in the tank
water. At the same time, a bacterial suspension of 106 cells ml−1 in PBS was added. During
the next 48 h, the flow of water was halted and then resumed. The bacterial treatment was
repeated every 24 h for 14 days, halting the flow of water for 6 h. The bacterial suspension
was replaced by the same volume of sterile PBS in the infection control tank, while neither
bacteria nor zoospores were added to the tank to control for death caused by ami momi
treatment. The fish were monitored under observation for 14 days to detect the emergence
of signs of saprolegniosis.

2.5.2. Biocontrol by Adding Bacteria to Feed

To conduct these experiments, three groups of 20 rainbow trout with an average body
weight of 29.29 ± 10.33 g were maintained in 40 L tanks: one tank for the bacterial treatment
and two tanks as controls. Fish were fed for 14 days with T2 Optiline 1P (Skretting® , Burgos,
Spain) feed containing 108 bacteria g−1 feed, with a daily administration of 2% of tank
biomass, followed by a fast of 48 h and 24 h before and after infection with S. parasitica,
respectively. After infection, the feed with the bacteria continued to be administered for
another 10 days. To prepare the feed with the probiotics, 10 mL of a bacterial suspension
with a concentration of 109 cells mL−1 was added to 90 g of feed, and the mixture was
homogenized with an electric hand mixer (HM 3100, Braun GmbH, Germany) for 1 min.
Subsequently, the feed was allowed to dry for 30 min at room temperature in a laminar
flow cabinet. Infection with S. parasitica zoospores was performed as described above, and
water flow was halted for the subsequent 24 h. The fish were maintained under observation
to detect the emergence of signs of saprolegniosis.
The origin of the fish, their acclimation, experimental conditions and the quality of
the physicochemical parameters of the water during these biocontrol tests were similar to
those detailed in Section 2.4.

2.5.3. Statistical Analysis

Survival analysis was performed by the Kaplan–Meier method, and the survival
probability of the groups treated or not with the probiotics was compared using the log-
rank test, considering that the differences were statistically significant at p ≤ 0.05. All
calculations were performed using SPSS for Windows version 26.

3. Results
3.1. In Vitro Inhibition Assays of the Probiotic Bacteria against S. parasitica
3.1.1. Assays with solid medium
The results of the plate assay in solid medium are shown in Table 2. On the negative
control plates, the colony of S. parasitica reached more than 5 cm in diameter (Figure 1). All
three bacteria showed a higher mycelial growth inhibition capacity when S. parasitica was
tested on BHI medium with bacterial strains previously grown for 7 days. L. plantarum
CLFP3 showed the highest fungistatic activity since it was already observed with a 3-day
bacterial culture.

Table 2. Inhibition of Saprolegnia parasitica in solid medium (plate assay) shown as mean ± standard
deviation (n = 3) colony diameter (cm) after culturing for 3 days at 20 ◦ C on a BHI plate with a 3-, 5-
or 7-day bacterial culture.

Incubation Time of
Enterococcus gallinarum L1 Vagococcus fluvialis L21 Lactobacillus plantarum CLFP3 Control Plate
the Bacteria (Days)
3 4.25 ± 0.05 5.45 ± 0.04 0 5.70 ± 0.11
5 3.46 ± 0.06 2.90 ± 0.05 0 5.17 ± 0.07
7 0 2.35 ± 0.04 0 5.65 ± 0.14
 or 7-day bacterial culture.

Incubation Time of the Enterococcus gallinarum Vagococcus fluvialis Lactobacillus plantarum

Control Plate
Bacteria (Days) L1 L21 CLFP3
3 4.25 ± 0.05 5.45 ± 0.04 0 5.70 ± 0.11
Animals 2023, 13,5954 3.46 ± 0.06 2.90 ± 0.05 0 7 of 15
5.17 ± 0.07
7 0 2.35 ± 0.04 0 5.65 ± 0.14

(a) (b)

(c) (d)

Figure 1.
Figure 1. In vitro growth inhibition
inhibition tests
tests of
of the bacterial
bacterial strains
strains against
against S.
S. parasitica
parasitica on
on BHIBHI agar
agar (a,b)
and in
and in 24-well
24-well tissue
tissue culture
culture plates
plateswith
withaacolonized
colonizedhemp
hempseed
seedand
andBHI
BHIbroth
broth(c,d):
(c,d): (a)
(a) S.
S. parasitica
parasitica
after 3 days of incubation at 20 °C
◦ on the control plate; (b) high level of inhibition of S. parasitica
after 3 days of incubation at 20 C on the control plate; (b) high level of inhibition of S. parasitica by by
Lactobacillus plantarum CLFP3 strain after 3 days (bacterial strain was previously grown for 7 days
Lactobacillus plantarum CLFP3 strain after 3 days (bacterial strain was previously grown for 7 days at
at 20 °C); (c) S. parasitica after 3 days of incubation at 20 °C used as a control well; (d) partial growth
20 ◦ C); (c) S. parasitica after 3 days of incubation at 20 ◦ C used as a control well;5(d) partial−1 growth
inhibition of S. parasitica produced by Enterococcus gallinarum L1 strain (2 × 10 5 cells mL −)1 after 3
inhibition
days at 20 of°C.S. parasitica produced by Enterococcus gallinarum L1 strain (2 × 10 cells mL ) after
3 days at 20 ◦ C.

3.1.2. Assays with Broth Medium (Hemp Seed Test and Cyst Test)
Both E. gallinarum L1 and V. fluvialis L21 isolates partially inhibited the growth of
the mycelium of S. parasitica with the dilution containing 2 × 104 bacteria mL−1 and with
higher concentrations (Figure 1). On the contrary, L. plantarum CLFP3 did not inhibit the
growth of the mycelium with any of the concentrations used.
E. gallinarum L1 and V. fluvialis L21 also inhibited cyst germination at concentrations
greater than 4 × 103 bacteria mL−1 , but L. plantarum CLFP3 only controlled germination
with the highest bacterial concentration of 4 × 105 mL−1 .

3.1.3. Fungicidal Effect of the Bacteria

The three isolates showed to be lethal for S. parasitica, but the effect varied according to
the concentration and incubation time of the bacteria. A lethal effect on the mycelium was
achieved at lower bacterial concentrations as the incubation time increased. Thus, with one
or two days of incubation, a lethal effect was observed only with the highest concentration
(2 × 105 bacteria mL−1 ), while with 3 days of incubation, a lethal effect was observed
up to the third dilution (2 × 103 bacteria mL−1 ). V. fluvialis L21 was the bacterium that
presented the greatest fungicidal effect. An atrophied and small mycelium was observed
after a period of incubation if a lethal effect was produced, while the mycelium grew and
produced zoospores on the control plate and on the plate with a non-lethal concentration
of bacteria.

3.1.4. Assays with Supernatants

Culture supernatants only inhibited mycelium growth when they were undiluted, as
well as in the case of E. gallinarum L1 with the first double dilution (1:2). Regarding the
inhibition of cyst germination, none of the supernatants of the three bacteria was inhibitory.

3.2. Adhesion to Skin Mucus of the Probiotic Bacteria and Inhibition of S. parasitica Cyst Adhesion
Table 3 summarizes the results for the adhesion of the three probiotic strains to
cutaneous mucus of brown trout. The adhesion was generally low (between 14.60% for
V. fluvialis L21 and 7.54% for L. plantarum CLFP3), but it was higher than the adhesion
of bacteria to bovine serum albumin and swine gastric mucus, although there were no
Animals 2023, 13, 954 8 of 15

significant differences between the adhesion of cysts to the mucus and that observed on the
other substrates (p > 0.05, Student’s t-test).

Table 3. Percentage of adhesion (mean ± SD of three experiments) of Enterococcus gallinarum L1,

Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3 strains to cutaneous mucus of brown trout
(CM), bovine serum albumin (BSA), mucin from swine stomach (MSS) and plate polystyrene (PP).

Bacterial Isolate CM BSA MSS PP

Enterococcus gallinarum L1 9.92 ± 7.59 4.57 ± 3.48 3.28 ± 3.48 4.39 ± 2.97
Vagococcus fluvialis L21 14.60 ± 5.48 7.87 ± 4.56 8.48 ± 3.89 7.98 ± 0.15
Lactobacillus plantarum CLFP3 7.54 ± 4.77 4.13 ±1.31 4.20 ± 1.45 4.51 ± 0.22

The results of the exclusion, competition and displacement of S. parasitica cysts are
shown in Table 4. In general, the three bacteria required lower concentrations to reduce
the adhesion of cysts in the competition test (bacterial isolates and cysts were added at the
same time). On the contrary, for the displacement of the cysts (bacterial isolates were added
after the cyst), higher bacterial concentrations were required. L. plantarum CLFP3 was the
bacterium that best reduced the adhesion of S. parasitica cysts in the three tests, despite
presenting lower percentages of adhesion to mucus than L1 and L21 isolates (Table 3).

Table 4. Percentage of reduction in the adhesion (mean ± SD of three experiments) of Saprolegnia

parasitica cysts to cutaneous mucus of brown trout tested with different bacterial strain concentra-
tions of Enterococcus gallinarum L1, Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3 under
conditions of exclusion, displacement and competition.

Bacterial
Bacterial Strain Exclusion * Displacement * Competition *
Concentration
2.5 × 107 28.55 ± 0.01 26.68 ± 2.92 65.03 ± 1.28
2.5 × 106 19.05 ± 6.73 24.93 ± 5.97 27.08 ± 3.05
Enterococcus gallinarum L1
2.5 × 105 7.50 ± 0.01 18.40 ± 14.77 6.33 ± 1.39
2.5 × 104 6.87 ± 1.89 0 6.26 ± 1.22
2.5 × 107 83.11 ± 1.31 93.24 ± 18.73 73.86 ± 5.55
2.5 × 106 62.81 ± 3.72 25.09 ± 9.21 40.03 ± 1.76
Vagococcus fluvialis L21
2.5 × 105 55.45 ± 35.84 13.59 ± 9.88 7.39 ± 3.14
2.5 × 104 17.67 ± 22.71 6.45 ± 4.13 6.49 ± 4.15
2.5 × 107 87.50 ± 0.01 33.32 ± 8.14 33.33 ± 2.09
2.5 × 106 76.01 ± 6.48 22.30 ± 8.37 7.78 ± 1.45
Lactobacillus plantarum CLFP3
2.5 × 105 70.98 ± 8.39 11.05 ± 8.33 5.44 ± 1.44
2.5 × 104 46.49 ± 17.63 0 7.29 ± 0.68
* Values in blod show the percentage of reduction in the adhesion of S. parasitica cysts significantly different
(p < 0.05, Student’s t-test) from the control (no bacteria added) determined by testing the minimum effective
numbers of bacteria.

3.3. Pathogenicity for Rainbow Trout

Both E. gallinarum L1 and V. fluvialis L21 isolates were non-pathogenic for rainbow
trout. No signs of disease or lesions were observed in any of the inoculated fish, and no
mortality occurred during the days that the fish were kept under observation.
In the microbiological study of the internal organs of rainbow trout, only the injected
strain was reisolated in two fish inoculated with E. gallinarum L1. In one of the fishes, which
was inoculated intramuscularly, it was reisolated from the kidney, and in the other, which
was injected intraperitoneally, it was detected in the spleen.

3.4. Biocontrol of Saprolegniosis by Adding the Bacteria to Water or Feed

None of the three probiotic strains, i.e., E. gallinarum L1, V. fluvialis L21 or L. plantarum
CLFP3, was able to prevent S. parasitica infection in rainbow trout when added to tank water
or administered through feed. Between 24 and 48 h after infection, the first macroscopic
 Animals 2023, 13, 954 9 of 15

S. parasitica lesions were observed, while the first deaths were noted on day 3 post infection,
reaching 100% mortality between days 6 and 9 post infection both in the groups treated
with the probiotic strains and in those not treated (Figure 2), with no statistically significant
Animals 2023, 13, 954 differences observed. In the control groups (not infected with S. parasitica), 100%10 survival
of 16
was observed, except in the experiment with the E. gallinarum L1 strain, in which one trout
died 24 h post infection, probably due to the ami momi treatment.

Figure 2. Kaplan–Meier survival curves in rainbow trout (Oncorhynchus mykiss) treated with the
probiotics
Figure strains Enterococcus
2. Kaplan–Meier gallinarum
survival curves in Vagococcus
L1,rainbow fluvialis
trout L21 and Lactobacillus
(Oncorhynchus plantarum
mykiss) treated withCLFP3
the
probiotics strains
administered Enterococcus
in water gallinarum
or in feed L1, Vagococcus
and infected fluvialis
with Saprolegnia L21 and Lactobacillus plantarum
parasitica.
CLFP3 administered in water or in feed and infected with Saprolegnia parasitica.
4. Discussion
Previous studies have shown that E. gallinarum L1, V. fluvialis L21 and L. plantarum
4. Discussion
CLFP3 can be
Previous used as
studies potential
have shownprobiotics against vibriosis
that E. gallinarum or lactococosis
L1, V. fluvialis L21 and for sea bass or
L. plantarum
CLFP3 can be used as potential probiotics against vibriosis or lactococosis for seacontrol
rainbow trout. In the present work, the possible utility of these bacteria in the bass orof
saprolegniosis was studied. For this purpose, both in vitro inhibition studies
rainbow trout. In the present work, the possible utility of these bacteria in the control and compe-
of
tition for binding sites against S. parasitica and in vivo tests with experimentally
saprolegniosis was studied. For this purpose, both in vitro inhibition studies and compe- infected
rainbow
tition trout were
for binding sitescarried
againstout.
S. parasitica and in vivo tests with experimentally infected
rainbow The three
trout bacteria
were out. the growth of S. parasitica in solid medium, with the most
inhibited
carried
pronounced inhibition in the case of L. plantarum CLFP3, since it appeared after three days
The three bacteria inhibited the growth of S. parasitica in solid medium, with the most
of incubation of the bacteria. The greatest inhibition was observed in bacterial cultures
pronounced inhibition in the case of L. plantarum CLFP3, since it appeared after three days
that already had 7 days of growth. Our results agree with those obtained by Hussein and
of incubation of the bacteria. The greatest inhibition was observed in bacterial cultures
that already had 7 days of growth. Our results agree with those obtained by Hussein and
Hatai [20], although they found differences depending on the culture medium used; they
observed the greatest inhibition when they used BHI agar with longer incubation times.
Regarding the inhibition of S. parasitica in liquid culture with hemp seed, the strains
Animals 2023, 13, 954 10 of 15

Hatai [20], although they found differences depending on the culture medium used; they
observed the greatest inhibition when they used BHI agar with longer incubation times.
Regarding the inhibition of S. parasitica in liquid culture with hemp seed, the strains
E. gallinarum L1 and V. fluvialis L21 prevented mycelial growth, while L. plantarum CLFP3
was unable to control it. However, the three bacteria inhibited the germination of cysts,
although L. plantarum CLFP3 required higher concentrations. In all cases, the concentration
of bacteria necessary to inhibit mycelial growth was greater than that required to prevent
cyst germination. These data agree with those obtained by Bly et al. [16], who found that
small amounts of different bacteria of the Pseudomonas genus prevented the germination
of cysts. Carbajal-González et al. [18] suggested that the need for lower concentrations
of bacteria to inhibit cysts may be due to the fact that a hyphal thallus already exists in
hemp seeds, while the cysts have to germinate and form the mycelium. Inhibition of cyst
germination in vitro could indicate that these bacteria hinder germination on the external
surface of the fish, thereby helping to control S. parasitica infection. In addition, the three
bacterial isolates were lethal to S. parasitica. This fungicidal effect is of interest since the
three bacteria are able to inactivate S. parasitica and help control infection, acting not only
as fungistatics.
None of the supernatants of the three bacteria inhibited the germination of S. parasitica
cysts, in agreement with [16]; however, in our case, the supernatants did stop the growth of
the preexisting mycelium in the hemp seed. The greater inhibition exhibited by E. gallinarum
L1 may be due to the fact that this bacterium produces lactic and acetic acid, as well as
small amounts of ethanol [31].
Epithelium surface colonization and adhesion, interfering with the pathogen’s ad-
hesion, are two positives for the selection of candidate probiotics [36]. Adhesion to the
cutaneous mucus of brown trout for the three bacterial strains tested in the present work
was found to be low. The limited adhesion of bacteria to fish mucus agrees with results
reported by other authors, with greater adhesion on the intestinal mucus of rainbow trout
and other fish species than on cutaneous mucus [37,38]. Comparing the adhesion reported
by Sorroza et al. [30,31] for E. gallinarum L1 and V. fluvialis L21 to the intestinal mucus of
various marine fish and to the cutaneous mucus of brown trout obtained in the present
study, both bacteria presented a higher percentage of adhesion to intestinal mucus. These
results agree with those of Bálcazar et al. [38] who, using three bacteria from the intesti-
nal microbiota of rainbow trout, observed a greater adhesion to intestinal mucus than to
cutaneous mucus. The greater adhesion of E. gallinarum L1 and V. fluvialis L21 bacteria to
intestinal mucus may be related to the origin of these bacteria, since they were isolated
from intestinal mucus; however, L. plantarum CLFP3, which was originally isolated from
cutaneous mucus, presented percentages of adherence to skin mucus lower than the other
two bacteria. Chabrillón et al. [37] indicated no specific adhesion of bacteria to a specific
host or substrate but that the adhesion capacity depends more on the strain. This is in
agreement with the data obtained in the present study for E. gallinarum L1, V. fluvialis
L21 and L. plantarum CLFP3, since there were no significant differences in adhesion to the
different substrates used, although adhesion to skin mucus was greatest.
Although adhesion to skin mucus was low, the three bacteria isolates significantly re-
duced adhesion of S. parasitica cysts, although higher bacterial concentrations were required
for displacement of S. parasitica cysts. This agrees with the results obtained by Carbajal-
González et al. [18], who indicated that the greater need for bacteria for displacement may
be due to a high adhesion of S. parasitica cysts to mucus, making it difficult for bacteria to
subsequently eliminate them. The lowest concentrations of bacteria to significantly reduce
attachment of S. parasitica cysts occurred under competitive conditions.
An essential characteristic of a potential probiotic is its safety and lack of pathogenicity.
It has previously been shown that L. plantarum CLFP3 is safe for rainbow trout [32] and
that E. gallinarum L1 and V. fluvialis L21 are safe for sea bass [30,31]. In the present study,
found that L1 and L21 are also non-pathogenic for rainbow trout. The genera Lactobacillus,
Enterococcus and Vagococcus are included in the group of lactic acid bacteria, which are
Animals 2023, 13, 954 11 of 15

mostly non-pathogenic and considered part of the commensal microbiota in the gut of
several fish species. Thus, González et al. [39] reported the presence of L. plantarum and
V. fluvialis as a part of the microbiota of several species of freshwater fish, including rain-
bow trout, and Petersen and Dalsgaard [40] isolated various Enterococcus spp., including
E. gallinarum, from the intestine of fish from integrated chicken-fish farms. However,
Osman et al. [41] found signs of septicemia in Nile tilapia (Oreochromis niloticus) associ-
ated with two isolates of E. gallinarum. In addition, other species of these genera, such as
V. salmoninarum, have been isolated from diseased fish and are capable of causing mortali-
ties up to 50% in rainbow trout farmed at low water temperature [42]. Regarding the genus
Enterococcus, three species (E. faecalis, E. faecium and E. hirae) have been associated with ente-
rococcosis/streptococcosis both in freshwater and marine fish [43,44]. Another Enterococcus
species, E. seriolicida, initially described as pathogenic for yellowtail (Seriola quinqueradiata)
in Japan [45] was later shown to be really identical to Lactococcus garvieae [46].
Although together, the three probiotic strains used in the present study achieved
acceptable results in vitro, inhibiting the growth of S. parasitica and decreasing the ability of
cysts to adhere to the cutaneous mucus, none of the three strains showed effective results
in vivo to prevent experimental saprolegniosis in rainbow trout either through the water or
administered in feed. Nevertheless, the beneficial effects against S. parasitica, even if limited
to in vitro responses, must be considered since the combination of these bacteria with
other strains could ensure a sufficient synergistic effect, with the capacity to prevent the
presentation of the disease. Gram et al. [47] pointed out that the selection and application of
probiotics must be tested for each individual host–pathogen combination and that in vivo
activity cannot be predicted based on in vitro testing. These authors found that a strain of
Pseudomonas fluorescens AH2 was strongly inhibitory against Vibrio anguillarum in vitro and
that it was also able to reduce mortality in rainbow trout against V anguillarum infection
via addition to the tank water [48]. Later, they found that this probiotic strain also inhibited
A. salmonicida in vitro but was unable to prevent furunculosis in salmon (Salmo salar) in an
in vivo model [47].
On the contrary, Nurhajati et al. [24] managed to prevent infection by S. parasitica in
catfish (Pangasius hypophthalamus) by adding different concentrations of L. plantarum FNCC
226 to the water and found that the effect was dose-dependent, with higher concentrations
of S. parasitica necessary to achieve inhibition. The best concentration treatment was
between 4.2 × 105 cfu mL−1 and 8.4 × 105 cfu mL−1 of L. plantarum FNCC 226, which can
inhibit infection with a concentration up 4 × 107 zoospores mL−1 of S. parasitica. In our
case, the concentration of L. plantarum CLFP3 used was very similar (106 bacteria mL−1 ),
but the concentration of zoospores was much lower (3 × 102 zoospores mL−1 ), although
the results are not necessarily comparable since a different experimental infection method
was used, as well as different species and sizes of fish.
In previous works by our research group using the same experimental conditions, we
observed that two Pseudomonas fluorescens strains (LE89 and LE141) were able to prevent
infection by S. parasitica in rainbow trout when added to tank water but not when administered
with feed [28]. Subsequently, we investigated the possible mechanisms of action of these
two isolates and verified that they seems to be related to activity of competitive exclusion
and to the production of some siderophores [29]. In addition, Lategan et al. [13,14] found
that administration of Aeromonas media strain A199 in water significantly decreased the
incidence of saprolegniosis in eel (Anguilla australis) and silver perch (Bidyanus bidyanus) and
that the substance associated with the inhibitory activity was indole production [34]. In
another study [49], the application of supernatant from a culture of a bacterium identified as
Burkholderia sp. Reduced the rate of infection by Saprolegnia sp. in grass carp (Ctenopharyngodon
idella). The substance involved with that antifungal activity was thermostable and was
identified as 2-pyrrolidone-5-carboxylic acid, a derivate of glutamic acid.
Competitive exclusion is generally considered a major strategy by which probiotic
bacteria prevent the adhesion of pathogenic microorganisms to fish mucous membranes.
Although in vitro studies have frequently reported this effect, studies with animals have
Animals 2023, 13, 954 12 of 15

failed to reproduce the same results with in vivo models on many occasions [50], which
could partly elucidate the results obtained in the present study.
Probiotics have been shown to have the capacity to increase innate and adaptive
immunity of fish, with the effects exerted on the fish innate immune system as the main
desirable characteristics [36]. Pérez-Sánchez et al. [51] investigated the expression of
immune-related genes in rainbow trout feeding with L. plantarum CLFP3 (106 CFU g−1 f
for 36 days) and found that mRNA levels of IL-10, IL-8 and IgT were significantly higher in
the L. plantarum group compared to the control group after Lactococcus garvieae infection,
suggesting that protection conferred by L. plantarum was mediated by the stimulation of
the immune response.
In the present study, the possible mechanism of action of the three isolates used was
not investigated, so we do not know whether these isolates could increase the immune
response of rainbow trout under the tested conditions. The mechanisms by which probiotics
stimulate the immune system are not yet well understood, but it is known that factors
such as the type of strain, dose, duration and mode of administration or environmental
conditions can affect the immunomodulating potency of probiotics [52]. In this work, a dose
of 106 bacteria mL−1 in a water tank or 108 bacteria g−1 for two weeks was used, which may
not have been sufficient to stimulate the immune system. However, in the studies in which
the usefulness of probiotic strains in the prevention of saprolegniosis has been confirmed,
their beneficial effect seemed to be related to the production of inhibitory substances or
competition for iron and not to the increase in the immune response [19,29,34,49].

5. Conclusions
The obtained results show that the use of an effective probiotic against a certain disease
in a host may not be effective against another pathogen or in another host and that the
results obtained in vitro may not always predict the effects when used in vivo. These results
highlight the importance of selecting the most appropriate probiotic and its mechanism of
action depending on the species of fish and the disease to be prevented.

Author Contributions: Conceptualization, J.-M.A.-G. and F.R.; Formal analysis, C.G.-P. and J.-M.A.-G.;
Funding acquisition, J.-M.A.-G. and F.R.; Investigation, C.G.-P. and J.-M.F.-G.; Methodology, C.G.-P. and
J.-M.F.-G.; Project administration, J.-M.A.-G. and F.R.; Resources, J.-M.A.-G.; Supervision, J.-M.F.-G. and
J.-M.A.-G.; Validation, J.-M.A.-G.; Writing—original draft, J.-M.F.-G.; Writing—review and editing, T.P.-S.,
D.P. and F.R. All authors have read and agreed to the published version of the manuscript.
Funding: This study was funded through research project AGL2014-54683-R of the Spanish “Ministe-
rio de Economía y Competitividad” and cofinanced through the European Regional Development
Fund (ERDF). C.G.P. was awarded a predoctoral contract by the “Consejería de Educación” of the
regional government “Junta de Castilla y León” cofinanced through the European Social Fund.
Institutional Review Board Statement: The animal study protocol was approved by the Subcom-
mittee for Experimentation and Animal Welfare of the University of León, Spain (Protocol num-
ber ULE_04_2015).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available upon request from the
corresponding author.
Acknowledgments: We are grateful for the assistance provided by the laboratory technician Gloria
Fernández–Bayón; the fish farm Los Leoneses in León, which supplied the rainbow trout; the “Servicio
Territorial de Medio Ambiente de León” of the “Junta de Castilla y León”, which supplied the brown
trout; and Skretting España S.A., which donated some of the feed.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
Animals 2023, 13, 954 13 of 15

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