1. Introduction
Nile tilapia (
Oreochromis niloticus) has been recognized as one of the most economically important freshwater aquaculture species in the last three decades, with a global market value of USD 7.9 billion in 2020 [
1]. Disease outbreaks have been regarded as the major obstacle to Nile tilapia farming worldwide [
1]. Fish pathogenic bacteria, especially
Streptococcus agalactiae, outbreaks cause high mortality rates in Nile tilapia farming with losses being reported up to 70% [
2]. The most common approach to inhibit and prevent pathogenic bacterial infections has been antibiotic administration. However, antibiotic usage and misuse leads to major concerns regarding antibiotic side effects. Misused antibiotics have stimulated a selection of antibiotic-resistant bacteria, escalating zoonotic risk in human health [
3]. Antibiotic residues in fish can also hamper international trade because most countries refuse to import of antibiotic-treated fish products [
4,
5]. To address the shortcomings due to antibiotic usage, probiotics are one promising alternative to prevent pathogenic bacterial infection in fish.
Probiotics are live microorganisms that can confer health benefits and protective immunity on hosts when administered in proper amounts [
6]. Probiotics possess several protective mechanisms against bacterial infections, including antimicrobial compound production, reduction of serum cholesterol, improvement of lactose metabolism, and prevention of colonization of pathogens by competitive inhibition. Probiotics can also stimulate mucosal barrier function and influence various aspects of the innate and acquired immune system by inducing phagocytosis and IgA secretion, modifying T lymphocyte response, enhancing T helper 1 response, and attenuating T helper 2 response [
7]. Additionally, probiotics can lower gut pH, release gut protective metabolites, and regulate intestinal motility and mucus production [
8].
Probiotic bacteria such as lactic acid bacteria (LAB),
Bifidobacterium, and
Bacillus spp. are recognized as the most common probiotic microorganisms. Several studies on the use of probiotic bacteria in aquaculture have indicated the capability for rendering protection against pathogenic bacteria infection, enhancing growth performance, and improving immune system in fish [
9]. Additionally, various studies reported that the screening and selection of probiotic bacteria from fish gastrointestinal (GI) tracts such as Nile tilapia [
9,
10] and Climbing perch (
Anabas testudineus) [
11] were successfully performed because fish GI tracts contain diversified, significant, and beneficial microbes that could be potential probiotics. Therefore, isolating probiotic microbes from fish GI tracts was an interesting approach to isolate probiotics against pathogenic infection in fish including Nile tilapia.
In addition to bacterial probiotics, yeasts have increasingly been regarded as potential candidates for probiotics. In the last few years, there has been an increase in research involving screening for novel yeast with probiotic properties. Several studies have demonstrated that many yeasts possess an ability to enhance growth performance, stimulate immune response, as well as protect and reduce mortality from bacterial infection in many fish species [
12]. Probiotic yeast strains have been isolated from varied sources such as fruit, fermented foods, dairy products, and fish GI tracts. Various studies have shown that yeasts isolated from GI tracts of fish, such as Mullet (
Mugil spp.) [
13], Salmonids, Corvina drum (
Cilus gilberti), and Great amberjack (
Seriola lalandi) [
12], demonstrated potential probiotic properties. In spite of their beneficial probiotic properties, isolation and screening of probiotic properties of yeasts have been largely focused on a few species [
14]. There have been few reports on the isolation of probiotic yeast research from GI tracts compared to probiotic bacterial isolation [
15].
Bacteria and yeast constitute a dominant part of the gut microbiome of fish [
14,
16]. The potential of these gut-isolated bacteria and yeast to display probiotic properties and antipathogenic bacterial infection represents a promising alternative for their potential application in developing of feed additives for Nile tilapia farming. However, the isolation of both probiotic bacteria and yeast from fish GI tracts has not been reported. In this work, we isolated both bacteria and yeast from fish GI tracts and evaluated their probiotic properties and inhibitory activities against Nile tilapia’s pathogenic bacteria. Following validation, our gut-isolated bacteria and yeasts demonstrated probiotic properties and antibacterial ability in different fish species. Hence, our probiotic bacteria and yeast uncovered from fish GI tracts represent a viable alternative for an application used in Nile tilapia fish farming and are transferable to other fish species to prevent the outbreak of pathogenic bacterial infection.
4. Discussion
On the basis of the host-specificity exhibited by members of autochthonous microbes and homologous hosts, we hypothesized that microbes readily isolated from fish GI tracts could be potentially applied as probiotic microbes to prevent pathogenic bacterial infection in fish farming. In this experiment, 242 microbial isolates, 66 bacteria and 176 yeasts were successfully isolated from fish GI tract samples from seven species, including Climbing perch, Striped snakehead, Common carp, Asian sea bass, Nile tilapia, and Soldier river barb. All the bacterial isolates were Gram-positive, non-spore forming, and catalase negative and were identified as LAB based on morphological and physiological characterization [
38]. Our yeast isolates were screened using a direct survival selection strategy under an extremely acidic environment. They were identified as acid tolerant probiotic yeasts that can be found in the GI tract of animals.
Antibacterial activity against pathogens is one of the key functional requirements for probiotic strain selection. Our results showed that 66 bacterial isolates and 15 yeast isolates demonstrated inhibitory effects against fish pathogenic bacteria. Previous studies also reported that
Lactococcus lactis [
10] and yeasts [
12,
13] isolated from the intestine of fish showed inhibitory effects against pathogens. Many reports indicated that the antibacterial effect manifested by LAB is due to the production of some antimicrobial compounds (AMCs) such as organic acids (lactic acid, acetic acid, etc.), hydrogen peroxide (H
2O
2), carbon dioxide (CO
2), diacetyl (2,3-butanedione), and bacteriocins [
39]. LAB produces organic acids as metabolites decreasing the intracellular pH which interrupts DNA and protein functions, leading to cell growth inhibition and eventually cell death. Moreover, LAB possesses the ability to produce either heat-stable or heat-sensitive proteins with antibacterial properties called bacteriocins [
40] that provoke the development of pores in the cell membrane of susceptible bacteria through which electrolytes in the cytoplasm leak out, thus leading to their death [
41]. In this study, both non-neutralized CFS and neutralized and boiled CFS were investigated on their antibacterial property. The non-neutralized CFS of LAB isolates demonstrated antimicrobial ability in an agar well diffusion assay. However, no inhibition zone was observed when the pH of the CFS was neutralized to 7.0 and subsequently boiled. This result indicated that the inhibitory activity was attributed to either secreted organic acids or possibly heat-sensitive bacteriocin. The antagonistic activity of the yeast isolates against the fish pathogenic bacteria might be attributed to the production of certain yeast products i.e., ethanol and antimicrobial compounds such as killer toxins or mycocins [
41]. In the present work, the results of the agar slab method showed that yeast isolates demonstrated growth-inhibiting properties against fish bacterial pathogens, but neither non-neutralized nor neutralized and boiled CFS exhibited antagonistic activity in an agar well diffusion assay. This might be because the antagonistic activity of the CFS was extremely weak or absent. The release of AMCs by microbes is influenced by many factors such as culture conditions, cell density, and population kinetics [
42]. In parallel to our study, Polak-Berecka et al. [
43] also suggest that the most potent antimicrobial activity was observed when the live cells of probiotics were used. Thus, the growth inhibition of fish pathogens was attributed to the live culture of yeast, not by yeast CFS.
The successful probiotic candidates with antagonistic effects against pathogens should tolerate high bile salt concentration and acidic pH in the fish GI tract. The common stomach pH value in Nile tilapia is very low (approximately 2.5) because of the presence of hydrochloric acid (HCl) activating the action of pepsin. In the present study, 39 probiotic bacteria isolates were selected to investigate these properties. In this research, the probiotic yeast isolates were isolated under an extremely acidic culture condition at pH 2.5; therefore, all yeast isolates were acid tolerant by default. Nine selected probiotic bacteria could survive at pH value 2.5 for 24 h. Many studies reported that potential probiotics such as
Lactobacillus spp. and
Enterococcus spp. isolated from freshwater fish could survive at pH 2 [
44,
45]. The most important pH homeostasis mechanism of LAB and some yeast species is the maintenance of intracellular pH by pumping protons (H
+) that result from extracellular acid dissociation processes via ATP hydrolysis using proton-translocating ATPase (H
+-ATPase) [
46,
47]. In addition, bile salt has strong toxic effects on the cell membrane of microbes [
48]. The concentration of bile salt in the fish GI tract ranged from 0.4 to 1.3% [
44]. The bacterial probiotics in this study demonstrated bile salt tolerance in the presence of 1% bile salt for 24 h, with poor (20 bacterial isolates) and medium growth (19 bacterial isolates). Under the same tested condition for bile salt tolerant assay, the 15 yeast isolates showed stronger tolerance against the bile salt than bacterial isolates. Our results are in agreement with previous studies reporting bile salt tolerance of many fish gut-isolated microbes [
10,
49]. The tolerance to bile salt of the LAB and some yeast strains is presumably related to the capability to create the bile salt hydrolase (BSH) enzyme that protects them against the toxicity of bile salts by hydrolysis of amide bond in bile salt molecules [
48]. Nevertheless, the valid mechanism of BSH of yeast has yet to be elucidated [
50].
Another important probiotic property is the ability to adhere to the GI tract mucous membrane, promoting the persistence of probiotics over long periods of time. The results of the probiotic adhesion assessment were obtained by absorbance of crystal violet dissolved from the cells attaching to the walls of the test tube. Among 54 selected probiotic isolates, 15 isolates of yeast and 39 isolates of bacteria exhibited adhesion property. Several studies showed the ability of probiotics to attach to the hydrophobic surface [
13,
51]. The probiotic adhesion depends on the cell surface hydrophobicity (CSH) affecting binding of probiotics to either biotic or abiotic hydrophobic surfaces e.g., glass, medical devices, and epithelial cells [
52]. For this reason, probiotics with adhesion to the glass surface might be able to adhere and colonize to the hydrophobic hydrocarbon of host intestinal epithelial cells. Furthermore, adhesion ability is a critical factor in allowing probiotics to localize and form biofilms rendering their colonization and safeguarding from unfavorable conditions of GI tract, which in turn limits the adherence and colonization of pathogenic strains on the intestinal mucous membrane [
53]. Biofilms are normally composed of microbial cells and secreted exopolysaccharide (EPS), which is essential for maintaining the stability of the biofilm structure. EPS produced by microbes has been detected by a change of colony color on CRA.
The six selected isolates which met the probiotic criteria were then subjected to the detection of survival rate under simulated GI fluid. To qualify as probiotics used in Nile tilapia, bacterial or yeast strains must survive for approximately 7 h in the gastric conditions of the stomach and reach the intestine alive in which they will exert their function because the estimated total digestion time of Nile tilapia is 7.15 h [
54]. Some selected probiotic bacteria and yeasts showed strong tolerance in gastric and intestinal conditions. These results are also in agreement with viability reported for other probiotics such as
Lactobacillus spp. [
55] and
Candida sp. [
56] in the presence of a simulated GI tract. HCl and digestive enzymes including pepsin and pancreatin in the GI tract can inhibit most microbes. With respect to resistance to pepsin, probiotic strains may convert the content of amino acids of their membrane proteins into non-hydrophobic amino acid which is not selectively hydrolyzed by the action of pepsin [
57]. Many researchers have reported an insignificant effect of pancreatic enzymes on intestinal microbes compared to bile salt [
58]. Notably, our yeast isolates (YCS1/1 and YON3/2) showed significantly higher survival rates than their control group after 7 h of incubation. These results indicate that bile salt possibly contributes to the growth of the yeasts by serving as a carbon and nitrogen source. Hernández-Gómez et al. [
50] reported that glycine or taurine amino acid released from conjugated bile salt when the amide bonds earn hydrolysis by the BSH could be used as carbon and nitrogen sources for growth by the intestinal microbiota.
According to safety considerations, hemolysis activity and antibiotic resistance were used to evaluate the safety of probiotic strains. The absence of β-hemolytic activity is regarded as a safety prerequisite for selecting probiotic strains [
59]. While the result of β-hemolysis is considered deleterious, γ-hemolysis and α-hemolysis are regarded to be safe [
60,
61]. In the current study, γ-hemolysis was shown by all selected yeast isolates, whereas α-hemolysis was the resultant acquired for all bacterial isolates. Diguță et al. [
62] also reported that all yeast strains with γ-hemolysis possessed valuable probiotic traits for aquaculture use. Similar observations of α-hemolytic probiotics were also reported in some LAB strains [
55,
63,
64].
To be regarded as having probiotic properties, a probiotic must not harbor acquired and transferable antibiotic-resistance genes [
65]. In this study, all of the bacterial isolates showed resistance to certain antibiotics used. As reported by Curragh and Collins [
66], antibiotic-resistant probiotics might have negative consequences because antibiotic-resistance genes might be transferable to pathogenic bacteria. Notwithstanding, the inherent resistance of LAB strains to some antibiotics was encoded by antibiotic resistance genes contained in chromosomes, and thus the transmission of antibiotic resistance would be much lower compared to ones in plasmid [
67,
68]. However, the beneficial effect of antibiotic-resistant bacterial probiotics is that probiotics can be co-administered with therapeutic antibiotics for treatment of an infectious disease [
69]. In our study, all yeast isolates (except YCS1/1) displayed resistance to all antibiotics. The resistance of the yeasts to antibiotics also enables them to be suitable for therapeutic application in patients undergoing antibiotic treatment [
62,
70].
The six selected isolates, namely CS1/3, CE1/1, AT8/5, YCS1/1, YCS1/3, and YON3/2, showed the highest similarity to
W. paramesenteroides,
W. paramesenteroides,
L. argentoratensis,
K. ohmeri,
K. ohmeri, and
C. tropicalis, respectively. Recently, studies of probiotic properties of
W. paramesenteroides isolated from fish such as Arapaima (
Arapaima gigas) [
71] were reported. However, it has not been formerly reported from Striped snakehead or Soldier river barb. To the best of our knowledge, this is the first study to report the isolation of culturable probiotic
W. paramesenteroides that was effective against several Nile tilapia pathogens from Striped snakehead and Soldier river barb.
L. argentoratensis comb. nov. was re-evaluated from
Lactobacillus argentoratensis or
Lactobacillus plantarum subsp.
argentoratensis by a combination of 16S rDNA sequence analysis and phylogenomic treeing [
72]. The characterization of probiotic properties of
L. argentoratensis and
L. plantarum subsp.
argentoratensis was rarely reported [
73]. Furthermore, the isolation of
L. argentoratensis from fish has not been previously reported. Accordingly, this is the first report of
L. argentoratensis isolated from Climbing perch which has exhibited probiotic properties and antibacterial activity against Nile tilapia pathogens. Even though yeast strains have also been reported to possess probiotic ability, few reports regarding probiotic yeasts are available. In the present study, two species of yeast,
K. ohmeri and
C. tropicalis, were found to be probiotics. The probiotic potential of these yeast strains was previously found [
74,
75,
76]. Although the isolation of these yeasts from several marine fish was reported [
77,
78], there is no record of isolation of these yeast strains from freshwater fish. Consequently, this study could potentially be the first record demonstrating antimicrobial activity against Nile tilapia pathogens and probiotic characteristics of
K. ohmeri and
C. tropicalis isolated from the GI tract of Striped snakehead and Nile tilapia, respectively. Collectively, probiotic bacteria AT8/5 (
L. argentoratensis) demonstrated an outstanding ability to inhibit all tested bacterial pathogens. Additionally, probiotic yeast YON3/2 (
C. tropicalis) can inhibit two species of tested pathogenic bacteria. Our isolated probiotic bacteria and yeast could be promising probiotics for application in Nile tilapia farming as feed additives to prevent bacterial infection.