In vitro selection of autochthonous bacterium with probiotic potential for the neotropical fish piauçu Megaleporinus microcephalus

[Seleção in vitro de bactéria autóctone com potencial probiótico para o peixe neotropical piauçu Megaleporinus macrocephalus]

ABSTRACT

The study aimed to isolate, identify, and apply in vitro tests on bacteria with autochthonous probiotic potential isolated from fifteen healthy specimens of Megaleporinus macrocephalus. The strains were selected from the intestinal tract of fish and inoculated in the Petri plate containing Sharp Man Rogosa Agar (MRS) for (48 hours at 35ºC). They were isolated based on a test of catalase, Gram stain, tolerance to different gradients NaCl (1, 2 and 3%), pH (4, 5, 6, 8 and 9) values and bile salts (2.5 and 5%), in addition to the inhibition zone against pathogens. Of the 42 strains isolated, ST1 and ST9 had higher values (p<0.05) for total viable cells (31.80±0.07 and 32.51±0.05 CFU/mL × 10⁸) respectively. In the resistance tests, strains ST1 and ST9 presented the best results, with emphasis on ST9 in the gradients of pH, high values of bile salts and larger inhibition zones against Aeromonas hydrophila and Aeromonas jandaei. The strains with the best results in the tests, ST1 and ST9, were identified by the MALDI-TOF-MS method as Enterococcus faecium. Thus, the isolated E. faecium bacteria, may be recommended as for probiotic use in farming the M. macrocephalus.

Keywords:
bacteria selection; lactic acid; inhibition of pathogens; specific species

RESUMO

O presente estudo visou isolar, identificar e aplicar testes in vitro em bactérias com potencial probiótico, autóctones, isoladas de 15 espécimes saudáveis de Megaleporinus macrocephalus. As cepas foram selecionadas do trato intestinal dos peixes e inoculadas em placas de Petri contendo Man Rogosa Sharped Agar (MRS), por 48 horas, a 35ºC. Foram isoladas com base em teste de catalase, coloração de Gram, tolerância a diferentes gradientes de NaCl (1, 2 e 3%), valores de pH (4, 5, 6, 8 e 9) e sais biliares (2,5 e 5%), além do halo de inibição contra patógenos. Das 42 cepas isoladas, ST1 e ST9 apresentaram maiores valores (P<0,05) para células viáveis totais (31,80±0,07 e 32,51±0,05 UFC/mL × 10⁸), respectivamente. Nos testes de resistência, as cepas ST1 e ST9 apresentaram os melhores resultados, com destaque para ST9 nos gradientes de pH, altos valores de sais biliares e maiores halos de inibição contra Aeromonas hydrophila e Aeromonas jandaei. As cepas com melhores resultados nos testes, ST1 e ST9, foram identificadas pelo método de MALDI-TOF-MS como Enterococcus faecium. Assim, a bactéria isolada Enterococcus faecium, pode ser recomendada para uso probiótico na criação do M. macrocephalus.

Palavras-chave:
seleção de bactérias; ácido lático; inibição de patógenos; espécie- específico

INTRODUCTION

Globally, aquaculture production is continuously expanding, generating approximately USD 250 billion in 2018 (The state…, 2020). However, intensive production has provoked the outbreak of diseases, mainly because of bacterial infection, causing productive and economic losses (Madani et al., 2018). For pathogen control, several antibiotics are used by fish farm, sometimes inappropriately (Doan et al., 2018), with deleterious effects on water quality parameters. In addition, chemicals can bioaccumulate in reared aquatic organisms and promote the selection of resistant bacteria (Qi et al., 2020).

As alternatives to chemical substances, probiotics are commonly used as a prophylactic management strategy, improving growth and immunology (Doan et al., 2018; Sousa et al., 2019). However, to obtain its benefits, the microorganisms with probiotic potential must completely colonize the intestinal tract of the host. Thus, autochthonous microorganisms commonly show greater colonization efficiency than allochthonous ones because of their specific relationship with the host (Dias et al., 2019; Sousa et al., 2019; Yamashita et al., 2020).

In vitro assays can aid in the selection of autochthonous bacteria with probiotic potential, determining their survival in different physiological conditions as well as their inhibitory capacity against pathogens (Dias et al., 2019; Pereira et al., 2019; Paixão et al., 2020; Qi et al., 2020; Sousa et al., 2019). Some studies have reported positive results for both in vitro and vivo assays using autochthonous probiotic bacteria in aquaculture, such as Lactobacillus spp. from lambari Astyanax bimaculatus (Jatobá et al., 2017), Bacillus cereus of tambaqui Colossoma macropomum (Dias et al., 2018), Enterococcus faecium of the species pirarucu Arapaima gigas (Sousa et al., 2019) and Lactococcus lactisa selected from jandiá Rhamdia quelen (Yamashita et al., 2020). However, despite several reports for aquaculture, some native fish species with economic importance remain without any scientific information about the use of autochthonous probiotics.

The Anostomidae family is the second most diverse among the Characiformes, with approximately 150 species (Fricke et al., 2019; Garavello and Britski, 2003). While the genus Leporinus comprises a little more than half of all the diversity of the family, with about 80 valid species (Burns et al., 2014 Ramirez et al., 2016). Recent attempts have distinguished phylogenetic differences in the monophyletic groups of species as distinct genera and have included 9 species of Leporinus in a new genus Megaleporinus, including macrocephalus (Birindelli et al., 2020; Ramirez et al., 2017). The genus Megaleporinus was diagnosed by the reduction in the number of teeth (only three teeth in each mandible), the presence of a ZW sex chromosome and most of them have a large body, reaching up to 500 mm LS (Ramirez et al., 2017).

Among the neotropical fish species with national importance, the native fish piauçu Megaleporinus macrocephalus of Paraná River Bay plays an important role in aquaculture (Garavello and Britski, 1988; Ramirez et al., 2017). It has been introduced into the Northern region of Brazil in state of Acre (Martins et al., 2017). The species shows well-developed reproduction in captivity (Martins and Yoshitoshi, 2003), a large growth potential (Takahashi et al., 2004), and readily accepts industrial feed, either extruded or pellet rations (Soares-Júnior et al., 2013).

Even with the large potential for captivity production, there are no reports about the use of autochthonous bacteria as probiotics for piauçu. Thus, the current study aimed to isolate and apply in vitro tests in autochthonous bacteria with probiotic potential for the neotropical fish species Megaleporinus macrocephalus.

MATERIALS AND METHODS

For the isolation of bacteria with probiotic potential, 15 M. macrocephalus specimens from extensive rearing (0.785±0.12kg and 26.59±0.23cm) were anesthetized (benzocaine 20mg/L), sterilized with 70% alcohol, and euthanized by medullar section according to protocols of the Ethical Committee for Animal Use (CEUA number 3991300420). Afterward, the intestinal tracts were removed, selecting the anterior and medium parts (approximately 1g), which were macerated in saline solution NaCl 0.65%, submitted to serial dilution (1:10 factor), and inoculated on petri plates containing de Man Rugosa Sharped Agar (MRS Agar) with 1% aniline blue. After inoculation, the plates were kept in an oven at 35ºC for 48 hours (Jatobá et al., 2008).

Only lactic acid bacteria with coccus and bacillus morphology, gram positive, catalase negative, and with a blue color were selected (Dias et al., 2019; Jatobá et al., 2008; Vieira et al., 2013). Developed blue colonies were isolated in petri plates containing MRS Agar (48 hours at 35ºC) through the streak plate technique to ensure the purity of the strain. To determine the bacterial growth kinetics, each strain was 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 (Jatobá et al., 2008). 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^-1). Based on these results, maximum growth rate and duplicating time of strains were calculated (Jatobá et al., 2008; Vieira et al., 2013).

In vitro assays were carried out with grown bacteria in MRS broth (24 hours at 35ºC) containing different levels of NaCl (0.0, 1.5, 3.0 and 4.5%), pH (4, 5, 6, 8, 9, and the control 7), and bile salt (2.5 and 5.0% w/v), all with four replicates (Jatobá et al., 2008; Vieira et al., 2013). Growth percentage was determined using absorbance at 63nm in a spectrophotometer (Jatobá et al., 2008; Vieira et al., 2013). The inhibitory ability against pathogens was evaluated measuring the inhibitory halo (Jatobá et al., 2008). Discs with a diameter of 0.8cm were removed from petri plates containing acid lactic bacteria and placed on petri plates containing Tryptone Soya Agar (TSA) previously inoculated with Aeromonas hydrophila, Aeromonas caviae, Aeromonas jandaei, Pseudomonas aeruginosa, and Streptococcus agalactiae. A positive control without probiotic bacteria, containing only antibiotic (oxytetracycline at 3 mg/L), was used to compare the results according to Vieira et al. (2013) and Paixão et al. (2020). After incubation (48 hours at 35ºC), the inhibition halo (mm) against the pathogen was determined. This experiment was performed in a completely randomized design with four replicates per treatment.

The bacterium with better performance regarding probiotic use was identified as the species level for method MALDI-TOF-MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) using the molecular weight of ribosomal proteins with laser shots at a wavelength of 260-337 nm. Scores ≥1.7 (Paixão et al., 2020; Sousa et al., 2019).

In vitro tests data and bacterial counts were square root-transformed and subjected to normality and homoscedasticity tests (Shapiro Wilk and Levene, respectively). Analysis of variance (ANOVA one-way) with post hoc Tukey´s test (p<0.05) was used to compare means, using the statistical software Past 3.0.

RESULTS

Of the 42 isolated strains, only 12 showed probiotic potential after the analysis of affinity biochemical characterization with aniline blue dye, Gram stain and catalase test. (Table 1).

The antagonistic capacity against pathogens was determined by the diameter of the inhibition discs; strain ST9 showed the best results (p < 0.05). This strain showed a greater inhibition halo against fish pathogens such as Aeromonas hydrophila and Aeromonas jandaei, followed by ST8 and ST10 with high values for Aeromonas caviae. Strain ST10 showed inhibition values like those for ST9 regarding Pseudomonas aeruginosa and Staphylococcus agalactiae. Both ST1 and ST2 demonstrated the lowest values for Staphylococcus agalactiae, followed by ST11 and ST12 against Aeromonas hydrophila. Strain ST9 also showed greater values when compared to the positive control regarding Aeromonas hydrophila and Aeromonas jandaei, but similar values for A. caviae, P. aeruginosa, and S. agalactiae (Table 3).

Table 1
Determination of the biochemical characteristics of the isolated piauçu (Megaleporinus macrocephalus) strains

Strain	Aniline blue dye	Gram stain	Catalase test
ST1	+	+	-
ST2	+	+	-
ST3	+	-	+
ST4	+	-	+
ST5	+	-	+
ST6	+	+	-
ST7	+	+	-
ST8	+	+	-
ST9	+	+	-
ST10	+	-	+
ST11	+	-	+
ST12	+	-	+

• * Affinity with aniline blue dye (+) or non-affinity (-); Gram stain: positive (+) or negative (-); catalase test: positive (+) or negative (-).

Two strains (ST1 and ST9) showed greater values (p<0.05) for total viable cells (31.80±0.07 and 32.51±0.05 CFU/mL × 10⁸) and lower duplication periods (4.39±0.04 and 4.36±0.04 h), respectively. The maximum growth rate was observed for ST1 and ST11 (0.15±0.01 and 0.15±0.02 cells/hour) respectively. In the resistance tests, strains ST1 and ST9 showed the best results over NaCl, pH, and bile salt variation, highlighting ST9 (p<0.05) for pH at alkaline levels (8) and high values of bile salt (2.5% w/v) (Table 2).

Table 2
Bacterial growth kinetics: total viable bacteria count after 24 hours (TVB - CFU/mL^-1 x 10⁸), maximum growth rate (MGR), duplicating time (DT); and tests of resistance to NaCl values, pH scales of bile salts (BS) in reducing the absorbance of strains (%), of autochthonous bacteria isolated from piauçu (Megaleporinus macrocephalus)

Strain	TVB (CFU/mL-1 x 108)	MGR (h-1)	DT (h)	NaCl 1%	NaCl 2%	NaCl 3%	pH 4
ST1	31.80±0.07 a	0.15±0.01 a	4.39±0.04 a	77.86±0.40 a	65.30±1.12 a	61.22±1.51 a	4.27±4.27c
ST2	5.09±0.01 c	0.07±0.02 d	5.69±0.02 e	74.55±0.70 a	55.95±1.49 b	37.48±2.04 b	3.20±1.9c
ST3	5.04±0.01 c	0.09±0.00 c	5.45±0.01 d	36.43±2.20 d	31.70±1.73 d	23.47±1.19 d	2.26±0.08c
ST4	4.87±0.00 d	0.04±0.00 e	6.01±0.02 f	47.97±0.91 b	39.88±1.25 c	22.91±0.51 de	2.94±0.89c
ST5	1.54±0.00 e	0.07±0.00 d	5.41±0.04 d	30.02±1.65 e	38.13±1.43 c	20.48±0.57 e	0.46±0.03d
ST6	4.93±0.06 d	0.06±0.01 d	5.28±0.15 d	26.92±0.77 f	22.32±0.44 g	13.78±0.47 f	0.37±0.05d
ST7	4.93±0.04 cd	0.07±0.00 d	5.00±0.06 c	40.30±0.81 c	25.26±1.35 f	15.74±0.57 f	6.30±0.45bc
ST8	1.53±0.03 e	0.06±0.01 d	5.44±0.14 d	35.80±2.33 d	22.91±0.51 fg	15.19±0.99 f	7.69±0.89b
ST9	32.51±0.05 a	0.10±0.00 b	4.36±0.04 a	74.76±1.20 a	67.74±0.76 a	62.76±0.93 a	26.22±0.93a
ST10	1.55±0.12 e	0.11±0.01 b	4.81±0.13 b	31.27±0.16 e	27.90±1.81 e	16.61±1.06 f	6.41±0.21bc
ST11	5.73±0.07 b	0.15±0.02 a	4.82±0.03 bc	44.73±0.38 b	37.26±0.20 c	26.37±1.66 c	0.19±0.08d
ST12	1.53±0.01 e	0.07±0.00 d	5.10±0.04 cd	31.07±1.69 e	28.90±0.79 e	15.49±0.46 f	8.62±0.54d
P-value	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001
	pH 5	pH6	pH8	pH9	SB 2.5%	SB 5%
ST1	21.41±1.68c	59.30±1.68c	65.89±0.74b	10.83±1.33a	48.69±2.78b	32.52±2.87bc
ST2	23.91±0.74bc	60.75±1.16c	75.28±1.55ab	7.99±0.33b	49.00±0.36b	23.66±1.22e
ST3	24.72±1.90bc	87.07±1.22a	71.07±1.63ab	7.82±1.33b	43.30±2.30bc	35.25±0.99b
ST4	23.66±1.40c	64.35±1.57c	37.65±1.67b	0.47±0.08de	43.09±0.95bc	29.45±1.20cd
ST5	10.93±1.34d	69.03±1.82bc	70.98±1.46ab	4.31±0.96c	41.15±1.44c	28.81±2.40d
ST6	9.33±1.27d	61.51±1.91c	35.48±1.40d	0.08±0.03e	29.74±3.16d	21.85±0.81ef
ST7	27.10±0.68b	61.69±0.93c	62.53±1.75d	1.11±0.46d	47.75±1.97b	32.39±1.26bc
ST8	37.49±2.24a	69.29±1.87bc	38.34±0.89d	0.57±1.15d	32.51±1.97d	20.09±0.46f
ST9	36.92±1.72a	76.37±1.56b	80.40±1.26ª	12.04±0.82a	80.78±0.44ª	51.25±0.56ª
ST10	29.19±1.14b	62.43±0.49bc	46.61±0.48cd	0.45±0.17de	44.92±3.72bc	23.94±0.20e
ST11	3.65±0.08e	62.26±1.13bc	52.95±1.83c	9.50±2.19ab	49.31±2.81b	29.02±0.50d
ST12	38.50±2.01a	66.83±0.72bc	41.12±1.16d	3.05±0.38c	25.03±2.07e	13.99±0.58g
P-value	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001

• Mean values (mm) ± standard deviation with different letter in the column means statistical difference by Tukey test (p<0.05).

Table 3
Inhibition halos of autochthonous lactic acid bacteria strains isolated from piauçu (Megaleporinus macrocephalus) against pathogenic bacteria Aeromonas hydrophila, Aeromonas jandaei, Aeromonas caviae, Pseudomonas aeroginosa, Staphylococcus agalactiae

Strain	A. hydrophila	A. jandaei	A. caviae	P. aeruginosa	S. agalactiae
ST1	10.90±0.34 d	11.85±0.70 e	11.53±0.46 bc	10.70±0.29 cd	8.35±0.31 d
ST2	10.98±1.23 d	10.23±0.44 f	10.05±0.70 c	9.45±0.41 d	8.20±0.14 d
ST3	10.83±0.36 d	11.58±0.46 ef	12.08±0.36 b	11.18±0.35 bc	8.43±0.30 d
ST4	10.80±1.19 d	14.00±0.54 d	12.45±1.22 b	11.00±0.36 c	9.20±0.32 d
ST5	11.45±0.33 d	12.05±0.44 e	10.12±0.42 c	10.85±0.35 cd	11.18±0.56 c
ST6	10.28±0.30 de	15.55±0.83 c	11.83±0.66 bc	11.88±0.94 bc	11.75±0.99 c
ST7	13.48±0.73 c	15.18±0.37 cd	12.58±0.43 b	11.95±0.90 bc	13.93±0.39 b
ST8	12.63±0.30 cd	13.60±1.13 d	14.98±0.90 a	12.53±0.86 b	13.98±0.87 b
ST9	19.40±0.99 a	19.08±0.79 a	15.18±0.56 a	15.10±0.84 a	16.45±0.89 a
ST10	11.58±0.46 d	15.78±0.46 c	15.65±0.30 a	13.93±0.52 a	15.03±0.95 ab
ST11	8.28±0.21 e	14.55±0.44 cd	12.83±0.68 b	12.03±0.59 bc	14.58±1.22 b
ST12	8.53±0.41 e	10.83±0.36 ef	12.68±1.04 b	11.48±0.41 bc	8.85±0.37 d
*Antib.	16.05±1.11 b	17.38±0.53 b	15.50±1.20 a	14.38±0.70 a	16.73±0.98 a
p-value	< 0.0001	< 0.0001	< 0.0001	< 0.0001	< 0.0001

• Mean values (mm) ± standard deviation with different letter in the column means statistical difference by Tukey test (p<0.05). *Antibiotic - positive control.

The strains were identified by the MALDI-TOF-MS method, with prevalence of the species Enterococcus faecium, Klebsiella pneumoniae, Edwardsiella tarda and at the genus level for Salmonella sp. (Table 4).

Table 4
Identification of autochthonous bacteria with probiotic potential isolated from the intestinal tract of piauçu (Megaleporinus macrocephalus) performed by the MALDI-TOF-MS method

Strain	Organism (Matched Pattern)	Score value	NCBI Identifier	Rank (quality)
ST1	Enterococcus faecium DSM 13589 DSM	1.61	1352	(-)
ST2	No Organism Identification Possible	1.43	_	_
ST3	Klebsiella pneumoniae RV_BA_03_B LBK	2.04	573	(+++)
ST4	Edwardsiella tarda CIP 68_6 CIP	2.07	636	(+++)
ST5	Edwardsiella tarda CIP 106473 CIP	2.03	636	(+++)
ST6	No Organism Identification Possible	1.39	_	_
ST7	No Organism Identification Possible	1.50	_	_
ST8	No Organism Identification Possible	1.44	_	_
ST9	Enterococcus faecium 20218_1 CHB	2.01	1352	(+++)
ST10	Salmonella sp (enterica st Dublin) Sa05_188 VAB	1.84	98360	(+)
ST11	Edwardsiella tarda CIP 103852 CIP	2.00	636	(+++)
ST12	Edwardsiella tarda CIP 68_6 CIP	1.96	636	(+)

• * Score 2.00 to 3.00 and Rank (quality) (+++): High-confidence identification; Score 1.70 to 1.99 and Rank (quality) (+): Low-confidence identification and Score 0.00 to 1.69 and Rank (quality) (-): No Organism Identification Possible. Second similarity method of MALDI-TOF-MS. GenBank: National Center for Biotechnology Information (NCBI).

DISCUSSION

Several studies have reported the benefits of the use of autochthonous lactic acid bacteria for aquaculture (Dias et al., 2018; Pereira et al., 2019; Jatobá et al., 2017; Sousa et al., 2019; Yamashita et al., 2020). Among the bacteria with probiotic potential, Enterococcus faecium stands out because of its ability to completely colonize the intestinal tract of the host when compared with heterotrophic bacteria and which is most likely related to its rapid growth rate in the intestine (Dias et al., 2019; Souza et al., 2019). In addition, it is a non-hemolytic species, does not harm the host (Dias et al., 2019), therefore, hemolytic activity is necessary to evaluate the probiotic and determine its infectivity in the host (El-Jeni et al., 2016).

Strains ST1 and ST9 showed higher growth rates, including higher viable cell numbers, when compared to the isolated strains of Pterophyllum scalare with 5 x 10⁸ CFU/mL (Dias et al., 2019); the adequate value for probiotic supplementation is between 10⁸ and 10⁹ CFU/g (ANVISA, 2017). Thus, probiotic bacteria can provide additional protection to intestinal mucus, probably forming barriers against pathogenic bacteria and improving the immunological system of the host (He et al., 2017; Souza et al., 2019).

However, the viability of colonization for probiotic bacteria depends on environmental conditions throughout the dietary supplementation and ingestion by the host (Dias et al., 2019). The bacterial growth rate undergoes alterations because of changes in chemical and osmotic aspects in the intestine (Erkkilä and Petäjä, 2000). In the scientific literature, E. faecium demonstrates large resistance, above 60% at an NaCl concentration of 3% (Dias et al., 2019; Paixão et al., 2020) corroborating the present values for piauçu.

Different levels of salinity can affect bacterial growth (Vieira et al., 2013). Fish have an ionic concentration to maintain their osmotic profile at the same level as the external environment, driving the energy usage for moments of stress (Weirich et al., 1992). Thus, chemical and osmotic aspects of the intestinal tract can influence the survival and colonization of probiotic bacteria, provoking cell membrane rupture (Erkkilä and Petäjä, 2000). The resistance of isolated bacteria to saline stress in vitro may be an indication of great intestinal viability (Vieira et al., 2013). Similar results have been observed for Lactobacillus plantarum isolated from Litopenaus vannamei (Vieira et al., 2013) and E. faecium isolated from P. scalare, with resistance at up to 3% salinity.

In this study, ST9 showed resistance to pH values from 4 to 8 and high values of salt bile at 5%, with survival rates above 50%. For this reason, acid and alkaline values would be used for the control of pathogenic bacteria (El-Jeni et al., 2016). The resistance reported for E. faecium in this study could be a factor to determine its probiotic potential for the host. In the scientific literature, some genera, such as Enterococcus, Bacillus, and Pseudomonas are described as resistant strains at pH 6, 9, and 7, respectively (Dias et al., 2019; Paixão et al., 2020; Qi et al., 2020).

In the intestine, bile salt acts in the emulsification of fat and some vitamins, but it can also break bacterial cell membranes (Lambert et al., 2008), affecting the levels of phospholipids and fatty acids (Vieira et al., 2013). Some bacteria are resistant to bile salt by using specific enzymes, thereby reducing the bactericidal effect (Erkkilä and Petäjä, 2000). A study with gene variants showed that the resistance of E. Faecium to the action of bile emulsion is related to variations in ion gradients by ATPase type V, which are in the membranes and function as proton pumps or sodium ions through an ion gradient, losing ATP (Senior, 1990). Resistance to bile salts was observed by studying the GltK gene and confirmed its deletion that sensitized E. faecium E1162 to the action of bile. The GltK can encode glutamate/aspartate protein permease in the transport system and therefore plays a role in resistance to bile (Zhang et al., 2013). Thus, resistance to high bile levels, observed for E. faecium isolated from M. macrocephalus, affirms the probiotic potential of this strain for dietary supplementation. For these reasons, resistance to factors such as stomach acidity, saline levels, and bile gradients promotes efficient intestinal colonization (Paixão et al., 2020; Vieira et al., 2013).

Inhibition ability against pathogens stands out as the most desired characteristic among the lactic acid bacteria used as probiotics in aquaculture (Dias et al., 2018; Sousa et al., 2019). Among the strains isolated from piauçu, ST9 E. faecium showed inhibition ability against Aeromonas hydrophila and Aeromonas jandaei, similar to the positive control with antibiotics. Inhibition ability against A. hydrophila, Pseudomonas aeruginosa, Enterococcus durans, Staphylococcus haemolyticus, Vibrio parahaemolyticus, and V. vulnificus has been reported for E. faecium (Dias et al., 2019; El-Jeni et al., 2016; Mao et al., 2020; Vieira et al., 2013). The genus Enterococcus contains some species with resistance to antimicrobial compounds and antibiotics through mutagenic processes (Pietro et al., 2016). Bacterial synergisms have been observed with co-cultivation of the probiotic strain of E. faecium CMGB16 added to the fractions of the culture of Bacillus cereus, whose effect on the strain Escherichia coli O28 was a greater susceptibility to the effects of the antibiotic, besides influencing its adherence patterns (Ditu et al., 2011).

Probiotic bacteria produce various compounds such as lactic acid, hydrogen peroxide, and bacteriocins to control pathogens, in addition to competing for specific space and binding sites in the intestinal lumen (Jatobá et al., 2017). Isolates of E. faecium strains showed enterokinase A and B compounds with anti-Listeria activity and high thermostability (Ghomrassi et al., 2016). Such characteristics make it attractive, in view of its probiotic potential in supplementing the species' diet, by its direct promotion of immunity and prevention of diseases in breeding. These results serve as a basis for future tests and applications in vivo.

The MALDI-TOF-MS analysis of the isolated M. macrocephalus strains identified three species, Enterococcus faecium, Klebsiella pneumoniae, Edwardsiella tarda and one of the genus Salmonella sp, with emphasis for ST1 and ST9 identified as E. faecium. However, the level of reliability and similarity of the analyses, highlighted the ST9 strain with a score of 2.01 as the most suitable candidate with probiotic potential for the piauçu. Thus, the diversity of bacteria isolated from the intestinal tract of piauçu reflects the variation of bacterial communities in the intestine of fish influenced by biotic factors such as host age, stage of development, intestinal structure, food, nutritional status and abiotic such as habitat, characteristics of water quality, competition and cultivation conditions (Ramirez and Romero, 2017; Roeselers et al., 2011; Salas-Leiva et al., 2017).

Dietary supplementation with E. faecium in fish promoted modulation of the immune system, associated with colonization of bacteria in the intestine, increasing mucus secretion and total proteins such as (immunoglobulin) and enzymatic activities (lysozymes) (Das et al., 2013; Lazado and Caipang, 2014; Van Doan et al., 2019). Furthermore, supplementation with E. faecium stimulates the increase in the number of defense cells such as intraepithelial T lymphocytes, production of antibodies (IgA), in addition to stimulating macrophages and dendritic cells in the production of compounds such as nitric oxide (Khalkhali and Mojgani, 2017).

Similar to the present study, enterobacteria were isolated from A. gigas, among them are Klebsiella pneumoniae and Edwardsiella tarda, they are gram-negative bacteria with pathogenic potential in aquaculture, highlighting the high resistance registered for k. pneumoniae against the tested antibiotics (Proietti-Junior et al., 2021. The K. pneumoniae bacterium isolated from a group of fish expressed three residence genes ESBL (bla SHV + bla CTX + bla TEM) against several tested antibiotics (Singh et al., 2017). Recently, studies carried out with tilapia have registered an accentuated 100% mortality of infected fish compared to the control group, without K. pneumoniae injection (Vaneci-Silva et al., 2022). Thus, the severity of the spread of this etiological agent and its degree of infection in fish are pointed out, causing a great economic impact.

The E. tarda is a versatile pathogen that can infect a wide range of hosts, from fish to humans (Li et al., 2012). It is a facultative and mobile Gram-negative bacterium, causing Edwardsiellosis disease, which can generate great economic losses in aquaculture (Lima et al., 2008; Woo and Bruno, 2010). A study carried out with isolates of Bacillus subtilis, Bacillus velezensis and Bacillus pumilus, significantly reduced the pathogenicity of E. tarda in zebrafish larvae increasing survival by 50%, and those infections may have occurred via the skin, gills and intestine, thus he observed promoting health through the use of probiotics (Santos et al., 2021).

K. pneumoniae and E. tarda, however the pathogenesis of bacteria of the genus Salmonella sp. are unknown in fish (Fernandes et al., 2018). They are facultative anaerobic bacteria, gram-negative and can to survive in different environments, including the aquatic one (Popoff; Le Minor, 2005; Oliveira and Vaz, 2018). The occurrence of this pathogen in fish can be transient and it is related to the management of creation, form of industrialization, inefficient hygiene practices, equipment and inadequate handling of food (Fernandes et al., 2018). Thus, in vitro isolation and selection protocols for autochthonous probiotic bacteria can help prophylactically in the prevention of diseases in aquaculture, and as an alternative to the use of antibiotics that favor and select increasingly resistant bacterial strains.

CONCLUSION

This is the first report on the autochthonous probiotic Enteroccus faecium isolated from Megaleporinus macrocephalus. The ST9 strain was considered the most resistant to the challenges of chemical gradients, in the simulation of physiological conditions and great inhibiting capacity against pathogens. These findings point to positive probiotic properties that should potentially be considered as a probiotic use in breeding the species in aquaculture.

ACKNOWLEDGMENTS

The authors thank the Coordination for the Improvement of Higher Education Personnel (CAPES) the National Council of Scientific and Technological Development (CNPq) for financial support to Fujimoto, R. Y. (304533/2019‐0).

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Publication Dates

• Publication in this collection
  30 May 2022
• Date of issue
  Mar-Apr 2022

History

• Received
  24 May 2021
• Accepted
  18 Feb 2022