1. Introduction
Aquaculture is one of the fastest-growing food sectors due to the high population demand for food and the decrease in natural fish stocks
[1]. This industry contributes 52% of fish for human consumption and 46% of the total livestock production
[2]. Sea bass production in Europe is estimated at 309,226 tons in 2022, and sea bass is one of the most important aquaculture species in Mediterranean countries, especially in Turkey, Greece, Egypt, and Spain
[3]. The production of European sea bass is carried out in almost all countries of the Mediterranean. During their first month of life, larvae feed on brine shrimp and rotifers. Afterwards, they begin to consume feed. There are different production methods: floating nurseries at sea, concrete tanks, or ponds on land. Commercial sizes range from 250 g to more than 2500 g. Normally, it takes between 20 and 24 months to reach 400 g from the time the larvae hatch from eggs
[3].
Nowadays, aquaculture tends to increase the amount of production to satisfy the food and animal protein human demand through high fish-stock density
[4]. To meet this demand, industrial and high-scale aquaculture has to solve many gaps. Overcrowding gives rise to the appearance of diseases due to the stress conditions that fish livestock experience
[5]. The main diseases in aquaculture farms are produced by bacteria, which cause great economic losses
[6][7]. Bacterial infections dominate the disease reports of European sea bass in the Mediterranean (75%). Reports confirmed
Vibrio spp.,
Photobacteria spp., and
Tenacibacillus spp. as the most frequent pathogens in European sea bass
[8]. In many cases, antibiotic treatment is beyond the reach of environmental and public health constraints. The administered antibiotics are absorbed at a certain rate, and the unabsorbed treatments go into the environment
[9][10] and could promote antibiotic-resistant bacteria
[11][12]. Multidrug-resistant bacteria are one of the greatest challenges in public health
[13][14].
For this reason, researchers have been looking into new alternative approaches such as probiotics. Probiotics, which comes from the Greek terms
pro and
bios, are “live micro-organisms which when administered in adequate amounts confer a health benefit to the host”
[15][16]. Based on this definition, researchers considered probiotics as live microalgae, live yeasts, and live bacteria that provide benefits to the host. The use of probiotics in aquaculture production is presented as an ecological, safe, and viable alternative to antibiotics
[17]. Moreover, the correct and effective use of probiotics can avoid great economic losses; although their production has certain costs at an industrial level, their application can generate economic benefits
[18].
The application of probiotic components on fish causes interactions with host intestinal bacteria. These interactions lead to the formation of a wide variety of metabolites, which could produce beneficial outcomes for the fish
[19]. Probiotics enhance host parameters such as growth or nutrient assimilation, immunomodulation, disease resistance, and survival rates and mitigate environmental stress
[20]. In addition, probiotics can modify the association between the host and microbe or even the microbial community. They also improve the utilization of feed by increasing its nutritional value and enhancing the host’s immune response against different pathogens. Commonly, the application of probiotics in fish industries has been administered via water or feed additives, either singly or in combination with other products or vectors
[21][22].
Thus, probiotics have been tested in aquaculture with diverse and interesting results.
2. Probiotic Modes of Action in European Sea Bass
Probiotics are an effective prophylactic treatment against different diseases in fish. Determining the mechanism of action by which a probiotic benefits the host is complex. The synergy between various modes of action and/or the interaction with different microbes may result in host benefit
[23]. In fact, some authors disagree on the correlation between in vitro and in vivo results. Tinh et al.
[24] elaborate an interesting review of the mechanisms of action such as colonization of the gut epithelium, production of inhibitory substances, competition for chemicals or available energy, nutritional contribution, green-water effect, interference with quorum sensing, and immunostimulatory function. Based on the large number of mechanisms that a probiotic can use to exert its action, to date, there is no complete agreement on the results obtained in vivo. Therefore, an increase in research is recommended by the research community to reinforce knowledge of how probiotics work
[24][25]. Among the several mechanisms used by probiotics in different microorganisms on European sea bass, the most common are the modulation of immune parameters, competitive exclusion for adhesion sites, production of inhibitory substances, and nutrient competition—digestion and enzymatic contribution (see
Figure 1).
Figure 1. Mechanisms of action of probiotics in European seabass. (1) Modulation of immune parameters—Host immune system responds to microbe-associated molecular patterns (MAMPs) present in probiotics, leading to different intracellular signaling cascades. (2) Competitive exclusion for adhesion sites—Inhibition of pathogen by the colonization of host tissues. (3) Production of inhibitory substances—Production of substances with inhibitory effects on pathogens by probiotics. (4) Nutrient competition (digestion and enzymatic contribution)—Use of nutrients by probiotics, preventing their use by pathogens. Modulation of digestive enzymes that could increase nutrient absorption and improve digestion. Production of beneficial enzymes for the host.
2.1. Modulation of Immune Parameters
The modulation of immune parameters by probiotic bacteria is diverse and complex. The immune system responds to pathogen-associated molecular patterns (PAMPs) present in pathogens. Pattern recognition receptors (PRRs), fundamental in the innate response, attract pathogens and bind to their PAMPs, triggering the activation of the innate immune response. The best-known PRRs are toll-like receptors (TLRs), which are transmembrane proteins expressed in different immune and non-immune cells
[26], one of which is toll-like receptor 2 (TLR2). Moreover, researchers have argued that probiotics possess microbe-associated molecular patterns (MAMPs) able to be detected by the host’s PRRs, triggering, after detection and binding, an intracellular signaling cascade leading to the expression of effector molecules such as cytokines
[27]. TLR2 has the capacity to recognize peptidoglycan, which is a main component of Gram-positive bacteria’s cell walls, including lactic acid bacteria (LAB) probiotics
[28]. TLR2 stimulation enhances the production of proinflammatory cytokines, such as IL-1β and TNF-α, and induces nitric oxide (NO) synthase. Also, TLR2 stimulation promotes the production of reactive oxygen species (ROS) and nitrogen species, essentials for mechanisms related to host antimicrobial defense. In addition, TLR2 activation has a crucial role in transepithelial resistance against pathogen bacteria
[29][30]. Thus, these operations enhance a host’s innate immune system in myriad ways such as increasing the production of lysozymes; enhancing phagocytosis and respiratory burst activity; and enhancing complement activity, peroxidase, antiprotease activity, and cytokine production
[2][31]. Moreover, some probiotic components contain specific receptors promoting the production of white blood cells (WBCs)
[32].
2.2. Competitive Exclusion for Adhesion Sites
Bacterial adhesion to host tissues is one of the mechanisms that pathogenic bacteria use to establish their infections
[33]. The action of probiotics, on many occasions, is to prevent this adhesion of pathogens, and this action can be specific due to the adhesion of probiotics to the pathogen or to its receptor molecules in epithelial cells or non-specific due to the presence of physicochemical agents
[17]. Passive and steric forces, lipoteichoic acids, electrostatic interactions, and specific structures such as external appendages covered by lectins can make this adhesion possible
[34]. Bacteria tend to compete with each other by the exclusion of or reduction in other species’ growth. The exclusion of adhesion sites is the main result of several mechanisms and properties of probiotic bacteria to suppress pathogen adhesion
[35]. This competitive exclusion of adhesion sites inhibits the action of pathogenic bacteria by blocking infection pathways
[36]. In fact, this ability to compete for the binding site with a pathogen is considered one of the main identification criteria for a probiotic
[23][34][37][38]. The interaction between surface proteins, produced by certain probiotic bacteria, and mucins creates specific properties that may inhibit the adhesion of pathogenic bacteria
[39]. Regarding European sea bass, the adhesion of probiotics (
Vagococcus fluvialis and
Bacillus velezensis) in intestinal mucus showed excellent results compared to a control
[40][41].
2.3. Production of Inhibitory Substances
The production of inhibitory substances is presented as an absolute advantage of probiotics
[42]. There is a wide range of inhibitory substances produced by probiotics. Siderophores, lysozymes, hydrogen peroxides, proteases, and antibacterial peptides—including organic acids, antimicrobial peptides, and bacteriocins—are all responsible for pathogen inhibition
[25][34][43]. The organic acids produced by LAB, mainly acetic acid and lactic acid, have the ability to penetrate pathogenic bacteria, reducing their intracellular pH or accumulating and causing the death of the pathogen. Therefore, they are considered the main probiotic antimicrobials against Gram-negative bacteria
[44]. In addition, two methods of bacteriocins-mediated pathogen clearance have been demonstrated: one includes cell wall perforation, and the other uses inhibition of cell wall synthesis
[45]. Regarding antimicrobial peptides, dicentracin is an antimicrobial peptide exclusively produced by European sea bass. Dicentracin has the ability to lysis a wide range of different pathogens, bacteria being the most known
[46][47]. The production of antimicrobial substances is not only directed against the lysis of the pathogen but also may be aimed at modifying the environment to make it less suitable for its competitors
[2][48]. Makridis et al.
[49] used
Phaeobacter sp. to improve the rearing of European sea bass larvae, showing an in vitro inhibitory effect against
Vibrio anguillarum.
Bacillus subtilis was tested in vitro against vibriosis in European sea bass larvae. Its supernatants presented a significant reduction in pathogen growth
[50]. In addition, previous research demonstrated the in vitro antagonistic capacity of
Vibrio lentus as a probiotic against six sea bass pathogens without pathogenic effects on European sea bass larvae
[51]. These facts might be attributed to the production of bacteriocins by probiotics. The same results were obtained by Öztürk and Esendal
[52], namely that the presence of
Lactobacillus rhamnosus through
Artemia nauplii considerably decreased
Vibrio spp. in European sea bass cultures. Additionally, El-Sayed et al.
[53] demonstrated the antibacterial effects of different probiotic microalgae in water against pathogenic bacteria. On the other hand, Monzón-Atienza et al.
[46] showed that the dietary administration of
B. velezensis D-18 enhanced the dicentracin gene expression. Also, Guardiola et al.
[54] showed different modifications of antimicrobial peptide gene expressions after
Shewanella putrefaciens Pdp11 supplementation.
2.4. Nutrient Competition: Digestion and Enzymatic Contribution
Nutrients are essential for bacterial growth. The use of similar nutrients gives rise to hostile competition among species
[55][56]. The utilization of available nutrients in environments by probiotics restricts their use by pathogenic microbes
[33][35]. In fact, this restriction resulting from competition for nutrients is one of the main mechanisms used by probiotics to inhibit pathogens
[43][57]. Iron is one of the most important nutrients for pathogenic bacteria since it is related not only to growth but also to virulence
[58][59]. For instance,
Bacillus spp. has shown a capacity to synthase siderophores and also has a higher organic carbon utilization
[60][61]. The absence of iron and carbon limits microbes’ pathogenic functions. Furthermore, probiotics have the capacity to release a wide range of digestive enzymes. Thus, an increase in digestive enzymes can lead to the degradation of nutrients
[62]. This digestive enzyme action can increase host nutrient absorption
[63]. Both probiotic actions limit the use of nutrients by pathogenic bacteria.
Several probiotics have been tested in European sea bass and have been observed to enhance the production of enzymes. For one, after the application of
Virgibacillus proomii and
Bacillus mojavensis, phosphatase alkaline and amylase presented higher values
[64]. Also, the simultaneous administration of
Lactobacillus farciminis and
Lactobacillus rhamnosus over 86 days upregulated acid phosphatase activity at day 8 and downregulated acid phosphatase activity at day 23 and a-amylase activity at days 8 and 103 post-administration. Furthermore, trypsin activity presented an increase from days 8 to 103
[65]. In reference to yeasts, various studies by Tovar-Ramírez et al.
[66][67] demonstrated the enzymatic modulation capacity of these probiotics in European sea bass. On the other hand, some authors have shown that the application of
Bacillus amyloliquefaciens for 42 days is capable of modifying the bacterial intestinal flora in European sea bass and reducing the presence of pathogens, surely due to competition for nutrients
[68].
In recent years, the study of how probiotics are related to the antioxidant response that occurs in the hosts has had a very important boom, carrying out studies to modulate the redox status of the host via their metal ion chelating ability, antioxidant systems, regulating signaling pathways, enzyme-producing ROS, and intestinal microbiota. The mechanisms of how they act are still not fully understood, and future studies are required to clarify the action of probiotics on the antioxidant response of the hosts
[69].
3. Probiotic Benefits in European Sea Bass Aquaculture
3.1. Increased Growth and Survival Rates
Probiotics in aquaculture promote fish growth by improving feed-conversion rates. The survival rate is another parameter that benefits after probiotic implementation [97]. As proof of these, many studies have reported that the application of different probiotics (single or combination) on European sea bass promote growth, growth performance, and survival [53–57,65].
3.2. Disease Resistance and Health Status
Like other species, European sea bass are susceptible to pathogen bacteria, viruses, fungi, and parasites
[70][71][72]. The application of probiotics in European sea bass has been shown to provide disease resistance. For instance, the administration of
Bacillus velezensis D-18 at 10
6 CFU/g over 30 days in European sea bass increased survival against
Vibrio anguillarum [46].
Bacillus velezensis also increased the cumulative survival rates against
Vibrio harvey SB
[51]. Similarly, the supplementation of
Phaeobacter sp. at 5 × 10
7 CFU/g in European sea bass fed via diets for 60 days increased resistance against
V. harveyi [49]. Sorroza et al.
[40] found a high survival rate against
Vibrio anguillarum after the application of
Vagococcus fluvialis at a high concentration (10
9 CFU/g) when compared with a control group. Likewise, both probiotic
Bacillus subtilis and
Lactobacillus plantarum at 10
6 CFU/mL demonstrated an increase in disease resistance in European sea bass against
Vibrio anguillarum [50]. In addition, the presence of
Pediococcus acidilactici in European sea bass increased survival against
Vibrio anguillarum [73].
In relation to the health status of the European sea bass after the administration of probiotics, different responses are affected, such as stress modulation, antioxidant status, hematological values, malformations, and parameters of the aquatic environment. Regarding stress, Lamari et al.
[74] showed the capacity of
Pediococcus acidilactici to downregulate HSP70 at 41 days post-hatching in European sea bass larvae. The HSP70 overexpression gene is considered a sign of improvement in acute stress resistance
[75]. Silvi et al.
[76] tested the effects of
Lactobacillus delbrueckii subsp. delbrueckii and found a stress decrease in treated European sea bass larvae. The same results were obtained by Carnevali et al.
[77] after the administration of
Lactobacillus delbrueckii subsp. delbrueckii in European sea bass, showing a decrease in cortisol levels. In addition, the application of
Vibrio lentus at four, six, and eight days post-hatching (dph) in European sea bass larvae had beneficial effects on stress by reducing glucocorticoids
[78].
Free radical formation occurs following different processes such as phagocytic activity as well as cellular metabolism
[79], which can lead to loss of biological function, tissue damage, and homeostatic imbalance
[80]. The formation of free radicals in fish occurs naturally after different metabolic processes
[79]. The presence of antioxidant substances is a fundamental factor in the elimination of free radicals. Antioxidants can be divided into enzymatic and non-enzymatic
[69]. It is well known that probiotics have the ability to produce enzymes or antioxidant substances or encourage the host to produce them
[79]. In fact, several studies have investigated the modification of the oxidative state after probiotic treatment in European sea bass. In one case, the presence of
Shewanella. putrefaciens Pdp11 in an experimental diet enhanced the oxidative status and the gene expression of superoxide dismutase (SOD) in European sea bass
[54]. Salem and Ibrahim
[81] also demonstrated that the sole application of
Bacillus subtilis HS1 decreased the levels of SOD, catalase (CAT), and total antioxidant capacity (TAC) in European sea bass. In contrast, the symbiotic application of that probiotic with chitosan enhanced SOD, CAT, and TAC. Furthermore, not only does the application of probiotic bacteria have these effects, but also the administration of live yeast—
Debaryomyces hansenii CBS 8339—showed a considerable decrease in antioxidant status
[82].
Regarding other health status parameters,
Vibrio lentus enhanced cell proliferation (haematopoiesis), iron transport, and cell adhesion in European sea bass larvae
[83].
Several authors have described the beneficial effects of probiotics in reducing malformations. In European sea bass, the combination of two different
Bacillus species—
Lactobacillus farciminis and
Lactobacillus rhamnosus—over 86 days at 10
8 CFU/g in feed considerably reduced malformations
[65] as well as the probiotic application of
Lactobacillus rhamnosus in European sea bass
[52]. Additionally, live
Debaryomyces hansenii reduced malformation appearance in European sea bass larvae
[66][67].
On the other hand, the surrounding medium is a fundamental factor in fish wellbeing, so water quality is considered an important parameter
[84]. Indeed, Eissa et al.
[85] demonstrated that the administration of
Pediococcus acidilactici in European sea bass culture improved water parameters and led to fish welfare as well as the application of live microalgae on water, which reduced the number of different pathogenic bacteria strains
[53].
3.3. Elevation of Immune Parameters
The application of probiotics enhances disease resistance by bolstering the immune system as well as general health. It has been demonstrated that probiotics improve different immune parameters in sea bass. In particular, non-specific immune parameters such as lysozyme activity, phagocytic activity, and respiratory burst as well as serum complement activity and the number of macrophages, lymphocytes, erythrocytes, and granulocytes have been modulated after the administration of probiotics in European sea bass
[46][54][83][86][87][88][89]. Furthermore, research has shown different modulations in cytokine levels after probiotic supplementation in European sea bass
[46][51][54][74][86][87][88][89]. In fish, an increase in immune parameters is usually related to higher survival rates. Several research studies of European sea bass have verified a high survival rate against pathogens after probiotic applications
[40][41][46][49][50][51][52][73][81].
3.4. Gut Morphology and Changes in Microbial Diversity
Symbiotic relationships between host and microbes are present in fish. Host and environment—biotic and abiotic factors, respectively—play a fundamental role in intestinal microbiota modulation
[90]. Microbes secrete metabolites, producing effects on intestinal environments and triggering changes in host physiology
[2]. Probiotics via intestinal–environment interactions may change host intestinal morphologies, thus increasing the surface absorption area localized in the mucosa and microbial diversity
[91]. That results in beneficial changes in host metabolism and energy expenditure
[92]. Changes in microbial diversity after probiotic supplementation have been related in European sea bass. Through denaturing gradient gel electrophoresis, Makridis et al.
[49] demonstrated an increase in bacterial diversity in European sea bass after the application of
Phaebacter sp. The dietary administration of
Bacillus amyloliquefaciens spores at 10
7 CFU/g had implications on gut morphology and microbial diversity in European sea bass. Previously, Silvi et al.
[76] showed that the application of
Lactobacillus delbrueckii subsp. delbrueckii in European sea bass modulated gut microbiota. Moreover, other studies have demonstrated an increase in the number of goblet cells, an increase in the villi length, and the absence of cyst formation, which is a clear indicator of an improvement in gut morphology. Also, after probiotic application, microbial diversity also benefited from a decrease in the Actinobacteria phylum and Nocardia genus. In addition, the number of Betaproteobacteria and Firmicutes—as beneficial bacteria—was higher
[68].