Comparing aquaculture to the terrestrial farms, based on pharmacological consumption, researchers have highlighted significant differences. Indeed, the World Health Organization classified aquaculture as an activity with a low environmental impact for antibiotic usage ^[1][2][7,8]. Directly linked to the above-explained concepts, veterinarians play key roles in pharmacological management. This consideration is justified because this professional figure firstly prescribes antibiotic therapies, avoiding the unnecessary administering (as metaphylactic one) of certain classes—in which usage is restricted to the human medicine: the so-called Critical Importance Antimicrobials WHO (CIA)—for food-producing animals; secondly, they must reduce administering to only restricted specific cases. The aim is to decrease a relevant selective pressure, which promotes resistant and pan-resistant bacterial strains survival ^[3][1]. These strains are named as antibiotic resistance bacteria (ARBs) that can be considered as “drivers” of antibiotic resistance genes (ARGs) with important repercussions on the environmental, animal, and human health ^[4][9]. It has been widely demonstrated that ARGs can be transferred to human intestinal microbiota and, consequently, to the ingestion of foods (numerous matrices: meat, dairy products, fish, etc.), which can drive commensal or pathogenic (Salmonella spp., Vibrio spp.) ARBs with extra chromosomal resistance forms. Finally, it is important to mention possible drugs residues due to the improper observation of legal limits ^[1][7]. Therefore, the European Regulations No. 470/2009 and No. 37/2010 established the residual limits of pharmacologically active substances in animal origin foodstuffs.

The finfish aquaculture zootechnic sector has been characterized by a wide range of farming techniques as the embankment ponds or the watershed ones (as observed in the catfish (Clarias spp., Ictalurus spp., and Pangasius spp.) culture) ^[5][65], mariculture systems (i.e., for Salmo spp., Sparus spp., ^[6][66], intensive or semi-intensive inland pond systems for Tilapia spp. ^[5][65]), and other animals finfish species, etc.

In the above-mentioned systems the high animal densities have induced the necessity of antibiotic administering for therapeutic purposes. This last-explained concept was associated with possible inappropriate usages and has selected resistant pathogens or commensal bacterial strains. More in detail, tetracyclines, beta-lactams, quinolones, and sulfonamides antibiotic classes have been largely prescribed by veterinarians. Therefore, biomolecular diagnostic procedures have coupled the next generation sequencing to the bacterial whole genome analysis. This last cited method has permitted reusearchers to discover new oligonucleotide resistance determinants ^[7][63]. Oligonucleotide sequences are the main actors involved in the ARGs circulation and are, consequently, responsible for the presence of vector bacteria (not usually resulting in pathogens for humans) while constituting crucial environmental reservoirs for the human and animal host microbiota ^[8][64]. For these reasons, animal origin foodstuffs have acquired more attention from scientists. The reasons were firstly related to possible residual concentration, but more specifically the main concern is represented by the possibility of horizontal resistance genes transmission between alimentary commensal and opportunistic strains with the human microbiota. Indeed, microorganisms have elaborated numerous mechanisms to disseminate the ability to survive by mobile genetic elements, such as integrons, plasmids, insertion sequences, transposons, and gene cassettes ^[8][9][64,67], and the inappropriate antibiotic usage has produced a selective pressure and the consequential survival of resistant microorganisms (engendering multiple resistances).

Every year, bacterial genome sequencing allows the identification of emerging and re-emerging ARGs, and the most frequent examples are amplified from aquaculture seafood products, i.e., sul (sulfonamides resistance genes), tet (tetracyclines resistance genes), aa (aminoglycosides resistance genes), and bla (β-lactams resistance genes) ^{[10][11][12][13]}[68,69,70,71]. Indeed, molecular biology, through the sequencing assays, constantly discovers different mutations among ARGs. There are numerous cases in which there is no matching between discovered phenotypic resistances results with the genotypic ones. This sentence offers explanations based on the concept of nucleotide sequences’ mutations that produce different DNA transcriptions (improper enzymes’ actions), or it is possibly correlated to an intrinsic resistance, which is typical of certain bacterial families against specific antibiotic molecules or classes. The scientific community, during these years, has investigated the AMR diffusion in various finfish species, especially in aquaculture systems, and the respective numerous genera of pathogenic and opportunistic bacteria that are generally implicated in seafood-borne diseases are Vibrio spp. (i.e., V. parahaemolyticus, V, vulnificus), Listeria monocytogenes, Clostridium botulinum, Aeromonas spp., Salmonella spp., Escherichia coli, Campylobacter jejuni, Shigella spp., Yersinia eneterocolitica, Bacillus cereus ^[14][41], Pseudomonas spp. ^[15][72], and Enterococcus faecium ^[16][73] (see Table 1). As previously mentioned, quinolones, tetracyclines, amphenicol, and sulfonamides are major antimicrobial classes used in aquaculture on a global scale ^[15][72].

Quinolone resistances are characterized by the involvement of DNA gyrase and topoisomerase IV, which are bacterial enzymes and quinolones target proteins. These two enzymes are encoded, respectively, by the gyrA and gyrB genes for DNA gyrase, and by the parC and parE genes for topoisomerase IV ^[29][38]. Chromosomal mutations in topoisomerases genes decrease drug accumulation and possible resistance driven by mobile elements, such as plasmid-mediated quinolone resistance (PMQR) (Qnr proteins, aac(6)-lb-cr aminoglycoside acetyltransferases and QepA and OqxAB efflux pumps), causing the constitutive or the acquired resistance to these antibiotic molecules. Increased mutations in DNA gyrase and topoisomerase IV, and in quinolone-resistant fish pathogens (Yersinia ruckeri, Flavobacterium psychrophilum, and V. anguillarum), are linked to the extensive administering of these antimicrobic classes worldwide ^[30][31][32][86,87,88]. Their wide usage was justified to reduce the hatching losses caused by Vibrio spp. infectious outbreaks. The wide detections of modified plasmids have been discovered from aquaculture finfish fillets ^[33][89]. The detected bacterial pathogens were A. hydrophila, V. anguillarum, and V. parahaemolyticus, which showed mutations in the quinolone resistance codifying sequences in specific gene regions belonging to gyrA and/or parC ^[34][35][90,91].

Quinolone resistance genes included in the so-called PMQR are: six qnr genes (qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC) encoding gyrase-protection repetitive peptides; oqxAB, qepA, and qaqBIII encoding efflux pumps; and aac(60)-Ib-cr encoding an aminoglycoside and quinolone inactivating acetyl-transferase ^[36][92]. The majority of PMQRs detection was largely amplified from finfish products worldwide; in China Yan et al. ^[37][93] found qepA and aac-(6′)-Ib genes as dominant among PMQR genes in aquatic environments and the possibility of co-emergence of resistance to β-lactams; Jiang and coworkers ^[38][94] detected qnrB, qnrS, and qnrD, with aac(6′)-Ib-cr in gut samples of farmed fish. Dobiasova et al. ^[39][95] found qnrS2, aac(6)-Ib-cr or qnrB17 genes in Aeromonas spp. isolated from tropical freshwater ornamental fish and coldwater ornamental (koi) carps. In Egypt, scientists reported the occurrence of qnr and aac(6)-Ib-cr resistance from fish farm water sample ^[3][1].

From an environmental perspective, there is a strict correlation between remarkable anthropic activities as polluted water areas and quinolones ARGs diffusion ^[40][99]. Indeed, in Asia (especially in China), fish farmers normally use biofertilizers to improve production ^[41][100]. There is a real possibility that these organic molecules are vectors of antibiotic resistance genes. Zhao et al. ^[42][101] examined biofertilizers normally used in Chinese shrimp aquaculture systems and studied the correlation between fluoroquinolone resistance genes’ diffusion and biofertilizers. In this research project, they also screened the PMQR gene that includes: qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB, and aaa(6′)-Ib genes. They screened 20 biofertilizer samples collected from shrimp farms and isolated 20 bacterial strains that were vectors of PMQR genes: 10 Escherichia coli, 9 Enterococcus faecalis, and 1 Enterococcus faecium. About 30% of biofertilizers samples presented qnrB, qnrD, and qepA resistance genes. This study was the first one which discovered the ARGs environmental repercussions due to the usage of contaminated manures on seafood farming systems. Similar patterns were also observed in terrestrial mammals, i.e., domestic swine and chicken manure (widely used in agriculture) ^[42][101]. Nowadays, there are not available data regarding finfishes, and it could be interesting to perform further investigations about possible statistical correlations between farming environments and possible agricultural implications. Therefore, these studies have confirmed ARGs diffusion and circulation in different environments through the fecal bacteria detected in common biofertilizer molecules. From these data, it can be seen that quinolones have presented reasonable risks due to the increase of their therapeutic failure. Their inclusion in the Critically Important Antimicrobials list has attracted more attention from pharma surveillance organizations.

Tetracyclines action consists of reversibly binding the 70S ribosome of cells blocking protein synthesis ^[3][1]. They are largely used in human and animal treatment as broad-spectrum antimicrobials. For the first time in Japan, it was observed that their improper administering conducted to the discovery of high nucleotide similarities of tetracycline genes between isolated bacteria from finfish aquaculture and from human clinical facilities. The phylogenetic analysis confirmed the same origin ^[43][3].

Evolution has selected different strategical and survival pathways and, in particular, four strategies: efflux pumps activation, ribosomal protection inducing a limit to the access, ribosomal RNA mutations avoiding tetracycline molecules binding, and tetracycline inactivation through enzymes ^[44][45][102,103]. In finfish aquaculture products, tet group responsible for proton-dependent efflux pumps encoding was mainly associated with tetracycline resistance ^[46][104]. Tet genes have been detected in several bacterial strains isolated from different animal species located in various geographical regions. There are multiple examples: tetB, tetM, tetW were firstly isolated in the intestine and rearing water of red seabream (Pagrus major) ^[47][105]; tetA, tetB, tetE, tetH, tetl, tet34, tet35 and 10 others had unknown tet genes isolated from Chilean salmon (Salmo salar) farms ^[48][106]; furthermore, Higuera-Llanten and coworkers ^[49][107] also detected the presence of tet34, tet35, tetA, tetB, tetE, tetH, tetL, and tetM genes in the same matrixes. Among seafoods (including finfish and crustaceans), Concha et al. ^[50][108] discovered tetX gene in Epilithonimonas strains from rainbow trout (Oncorhynchus mykiss) and Han et al. ^[51][109] amplified, in shrimp samples, that the tetB gene was carried in a single copy plasmid, named pTetB-VA1, comprising 5162-bp. The whole genome analysis revealed that this plasmid consists of 9 ORFs (overlapping open reading frames) encoding tetracycline-resistant repressor proteins, transcriptional regulatory proteins, and transposases and showed a 99% sequence identity to other tet gene plasmids (pIS04-68 and pAQU2). Furthermore, in terms of tet genes, with special regard to tetE, Agersø et al. ^[52][110] discovered tetE horizontal transmission between Aeromonas spp. and Escherichia coli strains, isolated from aquaculture Danish farms. TetA gene diffusion has been demonstrated to be realized through plasmids and transposons named Tn1721 and those that are Tn1721-like. Another similar example is represented by Tn5706, which is involved in tetH dissemination (amplified from Moraxella spp. and Acinetobacter spp. strains isolated from salmon farms) ^[53][111]. Due to the expanding of the AMR phenomenon among different bacterial strains, tet genes have been widely amplified from Enterobacteriaceae ^[54][55][112,113], Photobacterium spp., Vibrio spp., Alteromonas spp., Pseudomonas spp., and other marine commensal bacteria. Consequently, the possibility of transferring ARGs from marine microbiota to the human one is considered reasonable. Indeed, many biomolecular investigations have highlighted the possible cross-species ARGs transmission through the foodstuffs ingestion ^[44][56][102,114].

The wide oligonucleotide diversities, as described above, are expressions of the mass administering of tetracycline. Any mammalian zootechnic sectors (i.e., domestic swine) have improperly used this antibiotic class, inducing multiplication and genetic transmissions to the next generations of bacterial isolates (from pathogen to commensal strains, and vice versa).

In aquaculture, sulfonamides are commonly co-administered with trimethoprim, ormethoprim, and florfenicol ^[57][115]. The dihydropteroate synthase (DHPS) enzyme, in the folic acid pathway, represents the biochemical target reaction ^[56][114]. Sulfonamide’s resistance mechanisms derive from mutations in the chromosomal folP gene that provides varying degrees of trade-off between resistance and efficient folate synthesis, decreasing DHPS affinity for the antimicrobial molecule ^[56][114].

Among the discovered ARGs, four different sul gene determinants have been described to encode antibiotic resistance. Sul1 gene has been founded in class 1 integrons and linked to other resistance genes ^[58][116]; sul2 is associated with non-conjugative plasmids of the IncQ group and to large transmissible plasmids, such as pBP1 ^[59][117]. Sul3 is characterized in the Escherichia coli conjugative plasmid pVP440; sul4 gene has been recently mobilized and phylogenetic inference pinpoints its putative origin as part of the folate synthesis cluster in the Chloroflexi phylum ^[60][118]. All described ARGs have a common action, which is represented by the reduction in strategical bacterial structural expression. The transmembrane architectures are widely involved in the cyto-chemical interaction between strains and antibiotic molecules.

The genome and proteome analyses revealed that a gene cluster, containing a flavin-dependent monooxygenase and a flavin reductase, is highly upregulated in response to sulfonamides action, as reported by Kim et al. ^[61][119]. Indeed, the biochemical analysis showed that the two-components (belonging to the monooxygenase system) were key enzymes for the initial sulfonamides cleavage. It was observed that the co-expression of the two-component system in Escherichia coli conferred decreased susceptibility to sulfamethoxazole, indicating that the genes encoding drug inactivating enzymes are potential resistance determinants. Comparative genomic analysis revealed that this cluster gene, containing sulfonamide monooxygenase (renamed as sulX) and flavin reductase (sulR), is highly conserved in genomic islands. These ones are shared among sulfonamide-degrading Actinobacteria, all of which also contained sul1-carrying class 1 integrons ^[61][119].

Sulfonamide’s ARGs distribution has been widely found in numerous fish and environmental specimens, i.e., Muziasari and coworkers ^[62][120] discovered sul1, sul2, and intI1 genes detection in all analyzed samples and the dfrA1 gene in most samples in aquatic farm sediment in the Baltic Sea ^[63][39]. Domínguez et al. ^[64][121] detected sul1, sul2, class 1 integron-integrase gene intI1, dfrA1, dfrA12, and dfrA14 from a salmon farm in Chile and revealed the occurrence of transferable integrons and sul and dfr genes among sulfonamide- and/or trimethoprim-resistant bacteria, as amplified from Actinobacter spp., Bacillus spp., Proteus spp., and Pseudomanas spp. isolates ^[32][88].

These last considerations highlight that environmental stimuli can be responsible for increased or reduced ARGs transcriptions. The deduction leads to the consideration that in the AMR phenomenon, “the environment” plays a crucial role, while human and animal health are only “direct consequences”.

Thiamphenicol and florfenicol belong to the amphenicol antibiotic class and have been largely administered in aquaculture farms. Due to the possible chemical residual persistence in finfish muscular tissues, various studies have demonstrated possible sanitary implications on humans, animals, and environments ^[65][66][48,126].

Focusing on risk-based approach (in accordance with the EU Reg. No. 852/2004 and No. 37/2010), the European agencies EFSA and EMA published maximum residue limits and respective daily intakes for final human consumers ^[65][48].

Veterinary practitioners normally treat infectious disease (caused by, i.e., Vibrio spp., etc.) and relative possible septicemia cases using the above-mentioned molecules ^[67][127]. These have pharma-dynamic synergic effects (binding the 50S ribosomal subunit) if they were coupled with other antibiotic classes as tetracyclines. Both molecules have become widely prescribed because they have broad spectrum effects and low costs ^[68][128].

Amphenicol illegal administering has induced an intense evolutive pressure, determining the spreading of resistant strains harboring florfenicol-resistance genes (FRGs). These FRGs are plasmid determinants and have presented high genetic trades (through horizontal transmission) across different bacterial phyla, identifying strong correlations (p values < 0.05), as observed by Zeng et al. ^[67][127].

Among amplified FRGs, cat, cfr, cml, fexA, fexB, florB, and optrA have been discovered from animal origin food matrices (including finfish ones). Their biochemical actions are involved in several pathways, i.e., protein synthesis inhibition, exporter ability, methyltransferase activation, efflux pumps, etc. ^[69][70][129,130].

From a microbiological perspective applied to the veterinary clinical aspects, amphenicol administering has demonstrated biochemical repercussions on intestinal microbiota. It induces shifts among bacterial biodiversity acting as strong stressor ^[67][127].

This last consideration finds explanations from cyto-chemical interactions directly associated with the consequential expression of transmissible oligonucleotide sequences. Among the above-mentioned amphenicol-resistant determinants, the metagenomic technology, coupled with next generation sequencing, has identified multiple mutations on open reading frames regions, which encode resistant mechanisms, i.e., efflux pumps, new binding epitopes, etc. ^[66][126].

Innovative biomolecular technologies, combining thiamphenicol and florfenicol administering, has permitted reusearchers to reduce their respective dosages but preserve their therapeutic efficacy ^[71][131].

The notable ARGs heterogeneity and their extreme variabilities pose the basis for further diagnostic and One Health clinical challenges. Aminophenols, as with other previously mentioned antibiotic classes, are widely used in the aquaculture zootechnic sector. Therefore, it is mandatory to preserve their therapeutic actions.

FAO reports numerous administered vaccines against different bacterial or viral diseases among finfish species. The most frequently used provide seroconversion against Vibrio salmonicida, Vibrio anguillarum, Photobacterium damselae, Aeromonas salmonicida, Yersinia ruckeri, etc. ^[43][3].

Conversely, there are few vaccines for viral diseases, in which usage is highly recommended in marine finfish farms ^[72][132]. In the aquaculture farms, vaccines can be administered through different methods: injection, in bath, or through the orofecal route ^[43][3]. Injection, through the intraperitoneal route, provides powerful and durable protection, but, on the other hand, this procedure influences animal welfare, inducing a relevant stress condition. It is commonly used for Salmo salar finfish species but is not applicable for other species, i.e., Pangasius spp. and Tilapia spp. Conversely, oral administering reduces stress (due to animal handling), since animals receive immunization through food ingestion. The main difference between these two above-mentioned methods is represented by the need for large amounts of antigens in the ingestion method to obtain an adequate immunity ^[73][133]. There are contrasting opinions on vaccines’ efficacy regarding finfish farms. Usually, after vaccination, fish farmers must administer antibiotics to control infectious disease outbreaks ^[74][134]. This condition is related to an incomplete understanding of the vaccination type and the immune system’s reaction to the “antigenic stimuli”. It is improper to compare fish immune reactions with the generated response in mammalians ^[75][135].

However, in any species, such as farmed Atlantic salmons, vaccination represents an important preventive tool ^[43][3]. Farmed salmonids (Salmo salar) receive immune protection through the injection of a pentavalent vaccine against vibriosis, furunculosis, piscrickettsiosis, infectious pancreatic necrosis, and infection salmon anemia. The vaccine has permitted a reduced usage of antibiotics ^[75][135]. In tilapia’s farms, the mucosal administering route replaces the injective method. In this fish species, evidence supports a competent immune stimulation of the antigen-presenting cells (similarly to mammalians). In this way, fish farmers reduce antibiotic administering ^[76][136].

A new frontier is represented by nano-material vaccines, which use virus-like particles, immune-stimulating complexes, liposomes, polymeric, etc. These molecules drive antigens and can drive protective responses in fish. Furthermore, nanoparticles permit antigens’ release, and, for this reason, booster vaccinations are not necessary ^[77][137], but live attenuated vaccines’ employment in aquaculture is not allowed by the European Commission. Their usage has not yet been allowed due to the wide gap of knowledge concerning possible implications on human consumers ^[78][138].

Innovative production systems have become popular among fish farmers, i.e., catfish aquaculture in the USA ^[5][65]. New fish farming systems that provide more space and efficient wastewater management allow an avoidance of the large usage of antibiotic molecules ^[79][6].

Therefore, in the USA, fish farmers introduced an innovative system called “spilt-pond” to optimize fish’s sanitary conditions and productive levels. This new system is realized through the division of the traditional ponds in two areas: an algal growth basin and a fish holding area. In this way, the growth of production is allowed by the high animals’ density in the same period of production, and the reduction in antimicrobial use is due to the continuous water filtration ^[80][139]. Conversely, in Malaysia and other Asian-Pacific regions, fish are farmed by using pond culture, ex-mining pools, cement tanks, and freshwater pen culture systems. In these structures, there is low water filtration. Animal catabolites and feces remain for all productive cycles, producing a functional substrate for any bacterial species (i.e., Enterobacteriaceae) proliferation. Furthermore, in such countries of this continent (China, Vietnam, Philippines, India, etc.), the usage of antibiotics in aquaculture is not well regulated by national law ^[79][6]. Therefore, a new approach to the aquaculture systems of production is required. Indeed, Brunton et al. ^[81][140] generated a mapping system obtained through the stakeholders’ collaboration. Correlating ecological aspects to the new above-mentioned fish farms realities. It identifies hotspots and risk points related to antibiotic usage in the aquaculture food chain. The platform provides a quantitative risk analysis at different steps of production. Therefore, these maps allow researcherus to understand the molecules’ flows, ARGs, and ARB. In this way, it is possible to monitor antibiotic resistance factors. From these elaborated data, food safety authorities may program control activities through surveillance measures.

Probiotics have different effects on fish farming issues, i.e., they reduce animal mortality (especially at the larval stage) ^[82][83][84][141,142,143], improve animal welfare through the immune system’s stimulation, and reduce the antibiotic therapies’ necessity. Fish farmers introduce these bacteria through the finfish diet, as supplementary feed ^[43][3]. Bacillus spp. Is largely used in numerous fish farms realities for its probiotic properties. This genus can mitigate pathogenic microorganisms’ growth and can eliminate ARB ^[84][143]. These capacities are related to the bioactive peptides’ synthesis (bacteriocin) ^[85][144], but there are also nonpeptidic molecules, i.e., phospholipids, polyketides, etc., that are classified as bacteriocin ^[86][145]. Bacillus spp. produces CAMT2; this molecule is a recent example of bacteriocin that inhibits the proliferation of different bacterial strains, i.e., Vibrio spp., Staphylococcus aureus, and Listeria monocytogenes ^[87][146]. Another interesting microbiological aspect of this genus is represented by bacterial competition. It includes competition for energy (obtained from substrates), nutrients, and adhesion sites ^[88][5]. Indeed, Bacillus spp. Can rapidly colonize organic substrates with strong adhesion capacities (due to hydrophobic and steric forces) ^[89][147]. Therefore, pathogenic bacteria find an inadequate micro-environment that results in hostility to their proliferation. Furthermore, Bacillus spp. also stimulates fish’s cell-mediated immune response. Indeed, bacterial pathogens decrease their virulence because the animal host presents a resistant and competent immune system ^[90][148].

Healthy animals require few antibiotic therapies, leading to the reduction in antibiotic consumption in the next ten years ^[43][3]. Thanks to these preventive measures, different finfish species (i.e., parrotfish) have become resistant to Vibrio alginolyticus infection. These considerations are strictly related to the concept that powerful immune systems reduce pathogen bacterial proliferation ^[91][60].