In vitro bacterial probiotic selection from Nannostomus beckfordi, an Amazon ornamental fish

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Nonetheless, most of the probiotics applied in ornamental fish farming originate from allochthonous sources (Azevedo et al., 2016;Sousa et al., 2020;Paixão et al., 2020a) and sometimes have no beneficial effects on the host (Marengoni et al., 2010).Thus, the selection of bacteria specific to the fish (autochthonous bacteria) can provide greater colonization of the intestine and resistance against pathogens (Dias et al., 2019;Sousa et al., 2019;Yamashita et al., 2020).Thus, in vitro assay is extremely important in determining the bacterial resistance under physiological conditions (NaCl, pH and bile salts) and its capacity to inhibit pathogens (Dias et al., 2019;Paixão et al., 2020b).Based on in vitro tests, selecting bacteria strains with probiotic potential becomes easier and ensures benefits to the host (Dias et al., 2019;Sousa et al., 2019).Among the autochthonous probiotic bacteria, lactic acid bacteria, such as Lactobacillus plantarum, Enterococcus faecium, Lactococcus lactis and Weissella cibaria, were shown to benefit the fish (Mouriño et al., 2016;Jatobá et al., 2018;Dias et al., 2019;Yamashita et al., 2020;Paixão et al., 2020a).
Amazon ornamental fish have a high popularity in the national and international markets because of their attractive patterns and colors.Among the reared species, Nannostomus beckfordi (Lesbiasinidae family) is important and has a fusiform form, calm behavior, and red color on the fins (Weitzman and Weitzman, 2003;Abe et al., 2019).
There remains a gap in the scientific knowledge about the selection or even applicability of an autochthonous bacterium with probiotic potential for N. beckfordi.Therefore, this study aimed to isolate and select in vitro bacteria with probiotic potential for the Amazon ornamental fish N. beckfordi.

MATERIAL AND METHODS
To isolate lactic acid bacteria, twelve healthy fish (0.332±0.05g and 3.59±0.20cm)were caught (SISBIO 19515) in the Chumucui stream (01º12'38.3''S, 046º47'31.7" W).The fish were starved for 24 h and received anesthetic (bath with Benzocaine 60 mg/L) before euthanasia by medullar section according to protocols by the Ethical Committee on the use of Animal at the UFPA (CEUA -9202300420).Afterwards, they underwent a surgical procedure to remove the intestinal tract, homogenized in saline solution (NaCl 0.65%), conducted to serial dilution (1:10 factor) and then inoculated on a petri plate containing de Man Rugosa and Sharpe Agar (MRS Agar) with 1% aniline blue (1%).All plates were kept in a microbiological incubator for 48 hours at 35ºC (Mouriño et al., 2016;Paixão et al., 2020b).
The lactic acid bacteria selected (bacillus and coccus) with blue color, negative catalase and positive gram were inoculated in MRS broth and kept for 24 hours at 35ºC (Mouriño et al., 2016).To determine growth kinetics, the strains were inoculated in MRS broth and incubated for 24 hours at 35ºC.During incubation, an aliquot (3mL) was collected every 2 hours to determine absorbance 630 nm via a spectrophotometer.At the same time, another aliquot (100 µL) was inoculated on a petri plate containing MRS Agar and incubated for 48 hours at 35ºC to determine the colony-forming unit (CFU/mL).Based on these results, growth rate and final concentration were determined (FCU/mL) (Dias et al., 2019;Paixão et al., 2020b).
To determine the resistance under in vitro physiological conditions, an experiment in completely randomized design was conducted with different concentrations of NaCl (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%), pH (4, 5, 6, 8 and 9) and bile salt (5% w/v), all of them with three replicates and carried out simultaneously.The Falcon tubes were then incubated for 24 hours at 35ºC.Afterwards, using a spectrophotometer, the percentage reduction of absorbance at 630 nm was determined (Dias et al., 2019;Paixão et al., 2020b).
For the inhibition of pathogenic bacteria, the isolated strains previously inoculated on the petri plates containing MRS Agar were used to remove discs with a diameter of 0.8 cm.Each disc was placed on another petri plate containing Triptone Soya Agar with the pathogenic bacteria Aeromonas hydrophila (CPQBA22808 DRM), Pseudomonas aeroginosa (ATCC27853), Streptococcus agalactiae (LAQUA) and Aeromonas jandaei (LAQUA).This experiment was performed in a completely randomized design with tree replicates per treatment, after the incubation period (48 hours at 35ºC), inhibition halos (mm) were determined according to Dias et al. (2019).The bacterial strain with potential probiotic selected was identified used the method MALDI-TOF, using the molecular weight of ribosomal proteins with laser shots at a wavelength of 260-337 nm (Sousa et al., 2019;Paixão et al., 2020b).
All data were submitted to normality (Shapiro Wilk) and homoscedasticity (Levene's) tests.
Afterwards, all data were submitted to analysis of variance (ANOVA) with post hoc Tukey test (p<0.05).The percentage data were then submitted to transformation with arc sin root and microbiological counting with log (x+1).

DISCUSSION
The beneficial effects of probiotic supplementation have been reported for ornamental fish farming (Azevedo et al., 2016;Jinendiran et al., 2019;Sousa et al., 2020;Paixão et al., 2020a).However, there are few scientific reports about in vitro tests of autochthonous probiotics for Amazon ornamental fish.Currently, only P. scalare is reported in the scientific literature as an autochthonous probiotic (Dias et al., 2019).In the present study, E. faecium isolated from N. beckfordi showed greater values in resistance tests and a larger inhibition halo making it more efficient to colonize the intestine and promote modulating the gut microbiota, growth, and immune responses during phases in cultivating N. beckfordi.
For probiotic bacteria, colonization of the intestinal tract is the most important factor to promote the beneficial effects in the host (Sousa et al., 2019;Yamashita et al., 2020).Therefore, in vitro tests are necessary to select probiotic bacteria that present greater resistance to the host's physiological actions.(Paixão et al., 2020a;Khan et al., 2021).
In the fish, bile salt has an important function in emulsifying lipid during digestion and acting as a bactericide breaking the bacterial cell wall (Jahangiri et al., 2018;Brandvold et al., 2019), as observed for Lactobacillus plantarum isolated from Clownfish Amphiprion ocellaris (Paixão et al., 2020b).However, in the present study, E. faecium (strain 1) showed greater resistance to bile salt (growth above 60%) and a similar result was observed for E. faecium isolated from P. scalare (Dias et al., 2019).Thus, resistance to high bile levels observed for E. faecium isolated from N. beckfordi bare related to the gene expression of gltK that encodes the glutamate/aspartate permease, increasing bile resistance of probiotic bacteria (Zhang et al., 2013).
Sodium chloride concentration (NaCl) is another factor widely reported for selecting probiotic bacteria in vitro (Dias et al., 2019;Paixão et al., 2020b).Fish have an ionic concentration to maintain their osmotic profile at the same level as the external environment, and ionic changes in the digestive system of the host can affect the probiotic bacterium, causing rupture of the membrane (Moniruzzaman et al., 2018;Mortezaei et al., 2020).This hypothesis corroborates with the findings by Vieira et al. (2013), which found reduced growth for L. plantarum in the presence of 1.5% NaCl.For this study, E. faecium (strain 1) showed greater growth performance in 0.5 to 3.0% NaCl, an important characteristic of this probiotic bacterium for N. beckfordi, even if there were an osmotic imbalance in the specie, a similar result to E. faecium, Lactococcus lactis and Weissella oryzae (Dias et al., 2019;Mortezaei et al., 2020).
Bacterial resistance to different levels of pH has extreme importance for selecting a probiotic bacterium.In fish, the pH value can modify the intestinal microbiota, reducing the beneficial bacteria (Sylvain et al., 2016).According to Girijakumari et al. (2018), stomach acid can even reduce the bacterial load by 100%, affecting colonization (Sousa et al., 2019).In this study, all strains showed growth in acidic (pH 4) or alkaline medium (pH 9), showing better results with pH 5 and 6, mainly for S1 (E.faecium).Different results were observed in Dias et al. (2019) and Girijakumari et al. (2018) with P. scalare and Maylandia lombardoi, respectively, obtaining reduced growth at 100% for pH 5 and 2. In this study, the resistance of E. faecium can be a factor to determine its probiotic potential for N. beckfordi because the resistance of this probiotic bacteria to pH values allows the necessary amount in colony-forming units (CFU) for colonization in the intestine and promotes beneficial effects in the host.
The inhibition of pathogenic bacteria has been the most important benefit reported for probiotic diets in fish farming.Probiotic strains can release inhibitory compounds affecting the bacterial growth of pathogens, reducing fish diseases (Jinendiran et al., 2019;Yamashita et al., 2020).Among the strains isolated from N. beckfordi, the E. faecium (strain 1) showed an inhibition halo greater than 9 mm against four pathogenic bacteria, mainly for A. jandaei.This antagonistic characteristic observed for E. faecium in the present study can favor the resistance of the species to bacterial diseases during cultivation, thus avoiding possible outbreaks of mortality.
The antagonistic potential of E. faecium has been reported against A. hydrophila, P. aeruginosa, A. veronii, Staphylococcus haemolyticus, Vibrio parahaemolyticus, and V. vulnifcus (Dias et al., 2019;Mao et al., 2020;Paixão et al., 2020b).Its ability is related to the capacity to release lactic acid, hydrogen peroxide, and bactericides, antimicrobial compounds that control pathogens, in addition to competing for specific space and binding sites in the intestinal tract (Ng et al., 2020;Paixão et al., 2020b).Therefore, the use of E. faecium (strain 1) as a probiotic may be a strategy for the aquaculture of the Amazon ornamental fish species N. beckfordi.

CONCLUSION
This is the first report on the autochthonous bacteria E. faecium isolated from N. beckfordi.With adequate resistance under physiological conditions and greater inhibitory capacity against pathogens, it can be recommended as a potential probiotic for the aquaculture of this ornamental fish species.Aquaculture, v.518, p.734819, 2020.

Table 1 .
Values (Mean±S.D)of Maximum growth rate per hour (MGR/h), final concentration after growth at 24 hours (FCU/mL) and resistance tests (%) to different concentrations NaCl, pH and Bile Salt for isolated strains of the intestinal tract of N. beckfordi *Different letters in the same column indicate significant differences identified by Tukey test (5%).

Table 2 .
Values (Mean±S.D) of Inhibition halo (mm) of the probiotic strain isolated from the intestinal tract of N. beckfordi against pathogenic bacteria A. hydrophila (AH), A. jandaei (AJ), P. aeroginosa (PA) and S. agalactiae (SA) *Different letters in the same column indicate significant differences identified by Tukey test (5%).