Vibrio species are Gram-negative, asporogenous rods that are straight or curved, motile in aqueous environments usually by means of a single, polar flagellum (Kaysner et al., 2004). Vibrio spp. are mesophilic and chemoorganotrophic, possessing facultative fermentative metabolism (Kahla-Nakbi et al., 2007). They are ubiquitous inhabitants of aquatic environments including estuaries, marine coastal waters and sediments, and aquaculture settings (Balebona et al., 1998a; Thompson et al., 2004; Sarjito et al., 2009; Ringø, 2020). Except for V. cholerae and V. mimicus (Wong and Griffin, 2018), they are considered halophilic organisms (Wong and Griffin, 2018) commonly occurring at 30–35 ppt salinity although their aptitude to thrive in estuarine environments is also well-documented. The first Vibrio species described was Vibrio cholerae, in 1854, in the context of a study on cholera outbreaks in Florence (Thompson et al., 2004), but there are records of cholera-like diseases occurring in the times of Hippocrates (460–377 BC) (Blake, 1994). Currently, more than 130 species grouped in 14 clades in the Vibrio genus are recognized (Romalde et al., 2014; Huang et al., 2020), including commensal, mutualistic, and pathogenic species (Thompson et al., 2004).

The role of Vibrio spp. in marine organic carbon cycling (Romalde et al., 2014), particularly in coastal environments and marginal seas, has been underestimated (Zhang et al., 2018). Vibrio species are one of the best model marine heterotrophic bacterial groups, consuming several carbon compounds and growing with generation times as short as ~10 min (Zhang et al., 2018). They may represent about 60% of the total heterotrophic bacteria associated with aquatic organisms (Sonia and Lipton, 2012), being part of the normal microbiota of aquatic animals. Vibrio hosts are typically zooplankton, shellfish, crustaceans, benthic marine invertebrates such as sponges, corals and bryozoans, and fishes (Liu et al., 2016). Host-Vibrio relationships in nature may range from mutualistic through commensalistic to pathogenic (Liu et al., 2016). In general, Vibrio species proliferate well at warm temperatures, a condition that may favor their transition from commensal to pathogenic behavior. Environmental factors, including warming, have also been suggested to suppress fish immunity and increase their susceptibility to vibriosis (Haenen et al., 2014; El-Bouhy et al., 2016; El-Sayed et al., 2019).

In aquaculture, several Vibrio spp. are currently considered pathogens or opportunistic pathogens of reared finfish, shellfish, and shrimp (Liu et al., 2016). The most common Vibrionaceae spp. recorded in association with fish and shellfish diseases are V. anguillarum, V. ordalii, V. vulnificus, V. alginolyticus (Vera et al., 1991; Kahla-Nakbi et al., 2007; Korun and Timur, 2008), V. parahaemolyticus (Hamdan et al., 2016), Aliivibrio (formerly Vibrio; Urbanczyk et al., 2007) salmonicida, V. harveyi (Kahla-Nakbi et al., 2007; Korun and Timur, 2008), and V. tubiashii (Richards et al., 2014). Most of these species have been isolated both from reared and wild marine fish (Abdelaziz et al., 2017). Although V. cholerae is not referred to as a primary fish pathogen following Koch's postulates, it has been isolated from several freshwater and marine fish, which are considered a broad reservoir of V. cholerae strains that may cause infections in humans (Halpern and Izhaki, 2017). More recently, Devi et al. (2022) reported on a non-O1, non-O139 V. cholerae serotype (EMM1) capable of inducing high mortality in the freshwater species Labeo rohita, suggesting that V. cholerae strains other than the typical human pathogens shall be considered relevant aquatic pathogens as well. Vibriosis caused by the abovementioned species is the most common and devastating bacterial disease in fish larviculture and aquaculture, being a public health and economical concern affecting marine fishes, crustaceans, and bivalves worldwide (Balebona et al., 1998a; Sarjito et al., 2009; Ringø, 2020). The symptoms of vibriosis in fish are diverse, and include haemorrhagic septicaemias with extensive external skin lesions (haemorrhagic fins and ulcers), focal necrosis of some organs (liver, spleen, kidney), other tissue necrosis (Kahla-Nakbi et al., 2007), complete erosion of tail (Haldar et al., 2010), pale kidney, dark pigmentation, exophthalmic eyes, splenomegaly (Zorrilla et al., 2003), skeletal deformity (lordosis) (Abdel-Aziz et al., 2013), loss of appetite and lethargy (Korun and Timur, 2008).

Regarding the presence of Vibrio spp. in association with the live feed used for fish larviculture, several studies reported the isolation of the well-known causative agents of disease Vibrio alginolyticus (Yu et al., 1990), V. anguillarum (Dhert et al., 2001), V. parahaemolyticus (Balebona et al., 1998b) and V. rotiferianus (Gomez-Gil et al., 2003) from rotifers. The aquatic crustacean genus Artemia may also host Vibrio (Igarashi et al., 1989; Pousão-Ferreira, 2009), Pseudomonas (Igarashi et al., 1989) and Aeromonas (Interaminense et al., 2014) species. For instance, V. alginolyticus (Soto-Rodriguez et al., 2003; Interaminense et al., 2014), V. parahaemolyticus (Interaminense et al., 2014; Kumar et al., 2018), V. anguillarum (Campbell et al., 1993; Skjermo and Bergh, 2004), V. harveyi (Asok et al., 2012) and V. hispanicus (Gomez-Gil et al., 2004) have already been isolated from Artemia. Luminescent vibriosis (that is, vibriosis caused by luminescent Vibrio species) was as well-reported in Artemia and found to be caused mainly by V. harveyi and occasionally by V. splendidus (Soto-Rodriguez et al., 2003). Moreover, V. campbellii, frequently misidentified as V. harveyi in the past, was more recently found to be the etiological agent of luminescent vibriosis in shrimp hatcheries (Kumar et al., 2021).

Severe vibriosis in humans can be acquired by ingestion of contaminated water and raw or undercooked seafood (Wachsmuth et al., 1994; Finkelstein et al., 2002; Arab et al., 2020; Håkonsholm et al., 2020). Clinically, a few Vibrio species, despite their prevalently marine/estuarine origin, are able to elicit disease in humans. These include V. cholerae, V. parahaemolyticus, V. vulnificus (Wachsmuth et al., 1994; Finkelstein et al., 2002; Arab et al., 2020), V. alginolyticus (Gomathi et al., 2013; Citil et al., 2015), V. metschnikovii (Gomathi et al., 2013; Arab et al., 2020; Konechnyi et al., 2021), V. mimicus (Hernández-Robles et al., 2021), V. cincinnatiensis (Brayton et al., 1986), V. fluvialis (Ramamurthy et al., 2014; Kitaura et al., 2020), V. furnissi (Dalsgaard et al., 1997) and V. harveyi (Arab et al., 2020; Brehm et al., 2020). Well-documented symptoms of vibriosis in humans caused by Vibrio species which act as fish pathogens in aquaculture settings are highlighted below.

Vibrio alginolyticus had been frequently documented in early studies of gilthead seabream disease outbreaks in Mediterranean aquaculture (Balebona et al., 1998a). In humans, this bacterium was found to be associated with gastroenteritis in immunocompromised patients (Reina et al., 1995; Gomathi et al., 2013), causing extra-intestinal diseases (Gomez-Gil et al., 2003; Snoussi et al., 2008), wound infection, cellulitis, seawater-related otitis media (Abdel-Aziz et al., 2013; Gomathi et al., 2013), soft tissues and septicemia (Gomathi et al., 2013).

Vibrio parahaemolyticus is a well-known fish pathogen possessing a broad range of occurrence (Kumar et al., 2018). This bacterium was first recognized as a seafood borne pathogen to humans during an outbreak in 1950 in Osaka, Japan, involving 272 patients and causing the death of 20 people after the ingestion of Shirasu, a semi dried juvenile sardine (Aly et al., 2020). V. parahaemolyticus is the major food-borne pathogen worldwide (Bresee et al., 2002; Kawatsu et al., 2006), causing, after the ingestion of raw or undercooked seafood, acute dysentery and abdominal pain leading to diarrhea, nausea, vomiting, fever, chills, water-like stools, and an accentuated decrease of blood pressure leading to shock (Broberg et al., 2011; Siddique et al., 2021; Tan et al., 2021). In severe cases, patients become unconscious, with recurrent convulsions, becoming pale or cyanotic, eventually resulting in death. Antibiotic treatment and oral rehydration are the most common procedures to cure infections caused by V. parahaemolyticus. For individuals with critical physical or immunodeficiency diseases, the best practice to avoid severe illness is not to consume seafood at all (Wang et al., 2015). In the 21st century, increasing human disease outbreaks attributed to V. parahaemolyticus in Asia (Matsumoto et al., 2000), North America and Chile (Martinez-Urtaza et al., 2005), Europe namely France and Spain (Martinez-Urtaza et al., 2005; Quilici et al., 2005), Africa and Russia (Nair et al., 2007) have been described.

Vibrio vulnificus is highly pathogenic to humans (Snoussi et al., 2008). This bacterium causes epizootic outbreaks in seabream fish and can be transmitted to humans by ingestion, being a well-known cause of cellulitis and septicaemia in fishermen (Vinh et al., 2006). Besides, V. vulnificus is also able to infect the human host through an open cut or wound, in extreme cases resulting in necrotizing fasciitis, limb amputation and fatal septicaemia in susceptible individuals (Williams et al., 2014). The wound infections could start after the handling of infected fish and seafood, especially shellfish and after the practice of aquatic activities such as swimming (Hamdan et al., 2016; Baker-Austin and Oliver, 2018), being the consequences more severe when associated illnesses such as liver diseases, diabetes, and immune disorders are documented (Baker-Austin and Oliver, 2018). As usual among Vibrio spp., V. vulnificus possesses remarkable iron sequestration capabilities, meaning that the risk of infection is higher in humans with elevated iron levels (Wong and Griffin, 2018). More than 50% of primary septicaemia result in death within the first 72 h of hospitalization (Yun and Kim, 2018). Therefore, when there is a suspicion that the infection is caused by V. vulnificus, immediate and adequate antibiotic treatment and surgical interventions must be implemented. V. vulnificus is responsible for over 95% of deaths associated with seafood occurrences in the United States of America (Baker-Austin and Oliver, 2018). This is the highest fatality rate of any food-borne pathogen, which is in the range of category Biosafety Level 3 and 4 pathogens, namely anthrax, bubonic plague, Ebola, and Marburg fever (Baker-Austin and Oliver, 2018).

More recently, a few human infections caused by V. harveyi have as well been reported, underscoring the need of the public health sector to be aware of the possibility that wound infections caused by Vibrio species to humans may be becoming more likely to occur. With global warming, Vibrio-associated diseases will likely increase in the future (Brehm et al., 2020).

To avoid severe illness in humans caused by the ingestion of seafood contaminated with Vibrio species, thermal-based food processing such as low-temperature freezing (−18 or −24°C) or a 10 min high-temperature treatment (above 55°C) is common practice (Wang et al., 2015). Moreover, high-pressure processing and irradiation (using safety radioactive materials limits) are used to eliminate V. parahaemolyticus in oysters, keeping their original flavor (Wang et al., 2015). Notwithstanding the efficacy of hygiene measures employed in the preparation and processing of seafood for human consumption, devising novel, sustainable and green mechanisms of bacterial disease prevention in intensive fish farming holds promise in mitigating the impacts of the aquaculture industry on the environment and the risks posed to human health.

In 1997, the World Bank estimated that disease losses in aquaculture were worth US$3 billion per year, with Vibrio spp. having an important role in those losses (Laczka et al., 2014). Two decades later, estimates of disease losses duplicated (Stentiford et al., 2017). It has been suggested that all cultured marine fish around the world may, to varying degrees, host opportunistic vibrio species (Akayli and Timur, 2002), what does not necessarily imply that disease is always elicited nor that all vibrio species are pathogenic or will present pathogenic behavior. Yet multiple vibriosis outbreaks have been reported in several countries, infecting many fish species (Colorni et al., 1981; Akayli and Timur, 2002; Korun and Timur, 2008). In the case of the Mediterranean Sea, which is the primary habitat of gilthead seabream and a semi-closed water body, there is a limited rate of water exchange with open oceans. Aquatic pollution resulting from sewage, industrial effluents, crude oil refineries, and oil exploration affects the response of cultured fish to local environmental conditions (Guidetti et al., 2002). These factors also facilitate the invasion of bacterial pathogens (Vibrio, Streptococcus, Aeromonas, Pseudomonas) and parasites (nematodes, digeneans, acanthocephalans) into rearing systems. The ongoing chronic degradation of the Mediterranean Sea, thus, is considered to negatively impact the aquaculture industry in most of the North African coast (Eissa et al., 2017). For example, it has been suggested that the deterioration of water quality by sewage and agriculture discharges correlates with high prevalence of vibriosis in wild fish in the Mediterranean coast (Abdelaziz et al., 2017). Seawater exposed to higher anthropogenic pollution was found to display higher frequencies of Vibrio species, highlighting the importance of good manufacturing and hygiene practices to prevent and overcome fish vibriosis, even if innovative and “green” approaches were applied in industrial and domestic facilities (Abdelaziz et al., 2017; Arab et al., 2020).

The analysis of ten outbreaks involving Vibrio infections, affecting both cultured and wild gilthead seabream in the Mediterranean Sea (Table 1) revealed that V. alginolyticus was the Vibrio species most frequently isolated from gilthead seabream, followed by V. harveyi, V. splendidus, V. anguillarum, V. parahaemolyticus, and V. tubiashii. In these studies, Vibrio isolates were mainly recovered from seabream liver, spleen, and kidney, followed by external lesions and gills, but also from brain, eyes, gut, hepatopancreas, eroded tail and blood (Table 1). Interestingly, V. ichthyoenteri-like strains were isolated only from asymptomatic gilthead seabream individuals (Pujalte et al., 2003).

According to the Spanish outbreak (1990–1996) study performed by Balebona et al. (1998b), the species V. anguillarum, V. alginolyticus, V. harveyi, and V. splendidus were considered highly virulent for gilthead seabream by intraperitoneal inoculation, based on mean lethal dose (LD₅₀) values between 10⁴ and 10⁶ CFU per g body weight. In a further disease outbreak study, V. alginolyticus and V. harveyi were identified as virulent to gilthead seabream with LD₅₀ values between 10⁵ and 10⁶ CFU per g body weight (Kahla-Nakbi et al., 2007). In that study, V. alginolyticus and V. harveyi were isolated from the skin mucus of gilthead seabream, and no inhibitory effects of the skin mucus collected from gilthead seabream against those isolates was found. In fact, those Vibrio isolates showed remarkable serum resistance and were also able to adhere to skin mucus and grow using it as a nutrient source, suggesting high host colonization ability to eventually become an important infection risk.

Intriguingly, the etiological agents of human seafood-borne infections V. alginolyticus, V. cholera, and V. fluvialis were found in tissues of farmed gilthead seabream showing no disease symptoms (Arab et al., 2020), supporting the notion of a growing presence of the causing agent of cholera, V. cholerae, in farmed fish for human consumption (Halpern and Izhaki, 2017; Arab et al., 2020). Indeed, higher incidence of human pathogenic Vibrio species in coastal marine waters has been considered to result from climate change effects on the composition of marine microbial communities (Vezzulli et al., 2016). In this context, it is worth noting that vibriosis in cultured gilthead seabream has already been found to be induced by several factors such as transport stress, sudden temperature changes, low oxygen levels in water and handling procedures (Akayli and Timur, 2002). We posit that the trends observed in this review regarding gilthead seabream-Vibrio interactions are most likely applicable to a range of economically valuable fish species.

The continuous study of marine and estuarine microbiomes in coastal areas is of utmost relevance for a better understanding of long-term microbial community changes in highly productive ecosystems in the face of climate change. Such databases can guide the identification of beneficial microbes that can be used to mitigate the proliferation of opportunistic pathogens in immunocompromised hosts in built and open environments at large. Gilthead seabream and seabass are the main farmed species in the Mediterranean basin, and vibriosis was recently reported as the most common bacterial disease affecting these species (Muniesa et al., 2020). Based on the current literature, we argue that the occurrence of vibriosis in humans—and consequently the threats to human health posed by Vibrio species thriving in aquaculture settings—may be of a larger magnitude than previously thought. The frequency of human infections caused by estuarine and marine Vibrio spp. is likely to increase as ever-expanding intensive farming and global climate change synergistically interact to favor the proliferation of opportunistic microorganisms in livestock production systems (Reverter et al., 2020).

Species-level identification of members of the Vibrio genus, in an effective and standardized way, is necessary for a better bacteriological monitoring of farmed fish and the rearing environment within aquaculture facilities (Mustapha et al., 2013). As biochemical methods of identification often misidentify or are unsuccessful at the species level, molecular approaches must be implemented as common, accurate procedures for Vibrio species identification in seafood (Mustapha et al., 2013). However, it is important to note that, owing to the large genetic heterogeneity and fast diversification within Vibrio species, often 16S rRNA gene sequencing alone does not suffice for unequivocal identification of environmental strains at the species level. Bacterial species belonging to the Vibrio genus can differ in 16S rRNA gene nucleotide sequence from <1% up to 6% (Montieri et al., 2010). Particularly in the case of closely related species, the sequencing of a single marker gene may not be enough for precise taxon differentiation. For instance, V. parahaemolyticus and V. alginolyticus show quite similar biochemical properties (Mustapha et al., 2013) and are nearly identical with regards to 16S rRNA gene sequences (Montieri et al., 2010), prompting researchers to develop early DNA-based fingerprinting methods to discern between strains belonging to these species (Sadok et al., 2013). Indeed, V. alginolyticus was early designated V. parahaemolyticus biotype 2 (Aly et al., 2020), bearing testimony to the close phylogenetic relationship between these species.

Owing to the low discriminating power of highly conserved marker genes in distinguishing close Vibrio relatives, molecular identification based on species-specific markers and virulence genes have been considered adequate methods for species-level identification of Vibrio isolates (Mustapha et al., 2013). Because Vibrio spp. are symbiotic bacteria usually living in the intestine of aquatic species in a facultative way, genomic factors involved in the establishment of symbiosis and in the processes of host colonization and persistence may have evolved to confer adaptive advantage to species thriving in subtly different micro-niches. Several so-called “virulence factors”, such as enterotoxins, haemolysins, cytotoxins, proteases, lipases, phospholipases, siderophores, adhesive factors and/or haemagglutinins are produced by pathogenic species (Zhang and Austin, 2005). These traits allow Vibrio strains to adhere to the epithelial cells of fish juveniles, to break the first barrier of natural defense and to colonize all internal organs inducing vibriosis signs (Colorni et al., 1981; Paperna, 1984; Snoussi et al., 2008). It is important to note, however, that some of the abovementioned traits are common to several bacterial species and may likewise constitute adaptive features of mutualistic symbionts of fish (Borges et al., 2021). Overall, the use of genes coding for virulence or host-colonization factors as phylogenetic markers for the molecular detection of Vibrio species has gained increasing attention lately as nucleotide heterogeneities within such genes may reveal the adaptive behavior of different Vibrio species. A few PCR-based molecular identification systems of Vibrio species are listed in Table 2. These include protocols targeting genes coding for virulence factors which have been proved useful in discerning between closely related Vibrio species or in providing solid diagnosis of renowned pathogens, as reviewed more thoroughly in Supplementary File S1. For instance, several V. alginolyticus identification methods have been established based on the detection of hemolysin and collagenase encoding genes (Abdallah et al., 2011; Mustapha et al., 2013), and specific detection of V. parahaemolyticus and V. alginolyticus has been achieved through the exploration of nucleotide differences in genes encoding for the virulence regulatory proteins ToxR and ToxS (Abdallah et al., 2011; Aly et al., 2020). Also, a conserved virulence pathogenic island among Vibrio species has been exploited in the development of specific detection systems for the pathogen V. vulnificus (Table 2, see Supplementary File S1 for details).

It has been reasoned that the use of gene-targeted molecular tools may facilitate prevention of an outbreak as they allow the identification of the potential pathogens present even in asymptomatic fish (Altinok and Kurt, 2004). Yet it is presumably challenging to implement multiple gene amplicon sequencing methods in routine diagnostics for each different pathogen. In this regard, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an alternative technique often used in the identification of Vibrio species that may show advantages over PCR-based detection of phylogenetic marker genes. Indeed, MALDI-TOF MS is not labor-intensive, does not require highly trained operators, and is suitable for the processing of many samples in an automated, rapid, and cost-effective way (Li et al., 2018; Mougin et al., 2020). However, for accurate identification of closely related Vibrio species using MALDI-TOF MS, database choice is crucial (as in the case of species identification using phylogenetic marker genes). For instance, Moussa et al. (2021) found that correct discrimination of isolates belonging to the species V. tubiashii/V. europaeus and V. owensii/ V. jasicida/V. campbellii could not be achieved using some of the commonly available databases for MALDI-TOF MS-based classification. However, successful identification of diverse Vibrio isolates was achieved by Mougin et al. (2020) through the combined use of the Luvibase and Bruker v.9.0.0.0 databases. Thus, to fully exploit the potential of MALDI-TOF MS in fast and accurate identification of Vibrio species in aquaculture facilities, continuous development of comprehensive databases that allow discrimination between closely related Vibrio species is fundamental.

In conclusion, the current tools for fast identification of Vibrio pathogens in aquaculture facilities, or cultured fish, usually rely on the use of toxin-encoding genes, or other alternative functional marker genes, in targeted, PCR-based approaches (Abdallah et al., 2009, 2011; Aly et al., 2020) as well as on mass spectrometry protocols which have been gaining momentum in recent years (Mougin et al., 2020; Moussa et al., 2021). The combination of highly specific molecular identification of Vibrio pathogens, either by means of gene-targeted or mass spectrometry approaches, and broad characterization of total microbial communities via high-throughput 16S rRNA gene sequencing, for instance, is likely to become an effective approach to accurately determine the presence of opportunistic/pathogenic bacteria in complex microbial communities inhabiting aquaculture facilities, which may include beneficial bacteria with the ability to supress the spread of pathogens present in the community. The steady development of well-curated databases in support of molecular diagnostic tools will play a decisive role in enabling (i) the identification of multiple pathogenic agents present in a sample including understudied organisms, such as the likely emerging pathogenic species V. chagasii (Sanches-Fernandes et al., 2021b) and V. jasicida (Sanches-Fernandes et al., 2021c); (ii) targeting the specific group of pathogenic agents typical of each facility in a straightforward manner (Stentiford, 2017). Given that each aquaculture facility is unique, with singular and distinct features (Stickney, 2016), these approaches shall be used in a complementary way and considered in a case-by-case manner.

Infections caused by drug-resistant pathogens are responsible for 700,000 annual deaths in aquaculture, estimated to reach 10 million deaths as of 2050 (O'Neill, 2015). Recent studies suggest that antibiotic resistant bacteria may not only emerge in the environment due to the use of antimicrobial agents but also due to the increase of local temperature (MacFadden et al., 2018; Reverter et al., 2020; Pepi and Focardi, 2021), since it can affect bacterial cell physiology and promote mutagenesis, allowing antibiotic resistance mutations to take place early (Pepi and Focardi, 2021). Accordingly, the occurrence of Vibrio outbreaks is commonly higher during spring and summer seasons (Aly et al., 2020). Nevertheless, V. anguillarum was referred to as the etiological agent of fish vibriosis in both warm and cold waters in aquaculture facilities (Lages et al., 2019). There is, however, evidence that Vibrio–associated diseases are increasing in a global manner because of climate change and human activities (Vezzulli et al., 2016). This highlights the urgent need for more effective actions to combat not only the indiscriminate use of antimicrobial agents but also global climate change and warming (Reverter et al., 2020; Pepi and Focardi, 2021). The development of multi-resistance traits among pathogenic Vibrio spp. has been reported steadily across several aquaculture stations worldwide (Scarano et al., 2014; Aly et al., 2020; Deng et al., 2020; Dutta et al., 2021; see Supplementary Table S1), and it may be reasonable to argue that this trend results from the synergistic effects of past and currently unsupervised antibiotics use and higher water temperatures.

The common practice and overuse of antibiotic administration for prophylactic reasons in aquaculture is an important factor to consider regarding the increase in transfer of antibiotic resistance genes to land animals and human pathogens (Costa et al., 2015). This is a global public health concern exacerbated by the fact that the increment in multiple antibiotic resistance is also observed in food-borne pathogens, opportunistic pathogens, and the commensal microbiota of animals for human consumption, resulting in antibiotic resistance in the human gastrointestinal tract (Nguyen et al., 2014). With regards to the most crucial players involved in horizontal gene transfer among Vibrio cells, along with plasmids, phages, transposons and integrons are also genomic islands and integrating conjugative elements (Rodríguez-Blanco et al., 2012; Costa et al., 2015). The inheritance of resistance traits is often acquired via conjugation of resistance plasmids (R-plasmids), which commonly contain genes encoding resistance to multiple antibiotics. R-plasmids have been reported for Vibrio capable of transferring drug resistance traits such as V. alginolyticus (Gomathi et al., 2013).

Resistance profiles of Vibrio species isolated from diseased gilthead seabream (including the most important Vibrio pathogens of fish and humans, such as V. alginolyticus, V. harveyi, V. parahaemolyticus and V. vulnificus) toward antibiotics frequently used in the aquaculture industry are summarized in Supplementary Table S1. This meta-analysis reveals that antibiotic resistance profiles may vary among strains of the same Vibrio species or across studies of the same species, which is the case of the data gathered for Vibrio alginolyticus and V. harveyi. Furthermore, we observed that most Vibrio species are sensitive to tetracycline, oxytetracycline, chloramphenicol, and florfenicol. All studies listed in Supplementary Table S1 were performed in gilthead seabream rearing facilities in the Mediterranean zone. Collectively, the data suggest a trend for increased antibiotic resistance among diverse Vibrionaceae species at the Mediterranean basin, possible to observe across time for species such as V. aestuarius (from 1998 to 2014), V. alginolyticus (from 1998 to 2014, where studies published in 2007 and 2008 were performed in the same region by the same research group), A. fischeri (from 1998 to 2003), V. harveyi (from 1998 to 2020), V. parahaemolyticus (from 2013 to 2020) and V. splendidus (from 1998 to 2003).

A more responsible and prudent use of antibiotics in the aquaculture sector is important as they are present in all production stages. In 2011, oxytetracycline was the antibiotic with the highest prescription for both prophylactic and therapeutic ends in aquaculture facilities, and also the one that is most of the times freely available (Bondad-Reantaso, 2018). There is currently no international uniformization regarding antibiotics usage approval, which are licensed by each country in accordance with their own legislation (Guidi et al., 2018). For an overview of antibiotics currently in use in aquaculture and existing policies among major producing countries, we refer the reader to the recent review by Lulijwa et al. (2020). We list some of the most frequently used antibiotics in the aquaculture sector and in the Mediterranean area in Supplementary Table S2, whereby those antibiotics approved for use in Norway, Italy, Brazil, and the United States are disclosed. We observed that Vibrio species are sensitive to several antibiotics licensed for use (Supplementary Table S1), including oxytetracycline and florfenicol. While this picture is congruent with the need of applying effective measures to deter vibriosis outbreaks, it simultaneously raises concerns regarding the development of broader multidrug resistance traits among Vibrio species. Perhaps as important as delineating which antimicrobials are permissible in what quantities and where, surveillance of the fate of antibiotics in the environment and seafood biomass is key to ensure adherence of farming stations to local/national policies. In this regard, it is worth noting that the concentration of permitted antibiotics in seafood biomass often exceeds maximum residual limits in most of the major producing countries (Lulijwa et al., 2020). This calls for an urgent up-scaling of surveillance capabilities for better traceability and follow-up of antibiotic use practices (Schar et al., 2020).

The fact that antibiotics have been usually applied for prophylactic, therapeutic, and metaphylactic purposes favors the loss of susceptibility among the target organisms, and hence an increasing trend of antibiotic resistance among Vibrio species is likely as suggested by the data present in this review. Although several antibiotics have been banned or subjected to strict regulations, particularly in industrialized countries, the legacy effects of their past and current indiscriminate use turn the development of multidrug resistance among bacterial pathogens an important and timely public health concern (Pepi and Focardi, 2021).

The negative impact of the over usage of antibiotics on farmed fish species and coastal environments worldwide urges the development of alternative methods to prevent disease proliferation and reduce ecosystem deterioration caused by emerging, multi-resistant opportunistic bacteria. The development of strategies that target bacterial pathogens based on the activation of the host's immune system (i.e., using vaccines), on biological interactions such as pathogen predation (i.e., phage therapy) and on competition/niche displacement among microbes or beneficial host-microbe interactions (e.g., using probiotics), are gaining increasing attention because they may offer a less hazardous alternative regarding the suppression of fish pathogens in aquaculture.

For an overview on the use of vaccines to prevent fish diseases, we refer the reader to the comprehensive reviews of Embregts and Forlenza (2016) and Ma et al. (2019), the latter on the promises and challenges of oral vaccine administration. In short, vaccination programmes are considered an efficient, pathogen-specific suppressive approach that is best employed for disease prevention among adult fish, especially when injection methods of antigen delivery are adopted (Embregts and Forlenza, 2016). Indeed, the implementation of efficient vaccination programmes in Norway during the nineties is nowadays considered a remarkable example of how alternative disease control methods can sharply reduce the use of antibiotics in intensive fish farming (Lulijwa et al., 2020). Presently, commercial Vibrio vaccines such as AquaVac™Vibromax™ (Wongtavatchai et al., 2010) and ALPHA JECT 3000 (PHARMAQ AS, Norway; see Torres-Corral et al., 2021 for an example of application) are available, which offer efficacy in vibriosis control in shrimp and finfish, respectively, under different administration methods, namely incubation of Artemia nauplii prior to shrimp feeding (AquaVac^TMVibromax^TM; see Amatul-Samahah et al., 2020 for a review on vaccination of shrimp against vibriosis) and intraperitoneal injection of adult fish (AlphaJect 3000).

To improve fish wellbeing under vaccination programmes, oral administration methods using several modes of antigen encapsulation in delivery systems such as chitosan, alginates, and fish live feed such as Artemia and rotifers (bioencapsulation methods) have been attempted. However, improvements are still needed to ensure efficacy in antigen delivery in comparison with injection procedures. The main challenges of oral vaccine administration using encapsulation methods are to assure that vaccines reach the digestive tract of fish by ingestion, the maximum dosage allowed, which is dependent on the daily live feed intake, the time of exposure to be effective and the farmed fish tolerance to the vaccine (Embregts and Forlenza, 2016). The use of vaccines has moreover been considered not applicable to handle fish larvae and bivalves due to the lack of an adaptive immune system (Bentzon-Tilia et al., 2016), prompting researchers to consider alternative routes for disease prevention, such as the use of probiotics (see sub-section The Promise of Probiotics in Controlling Vibrio Diseases in Aquaculture).

Concerning phage therapy methods to prevent bacterial proliferation in aquaculture, the review by Richards (2014) covers pioneering studies on diverse bacteriophage-bacterial host systems and the efficacy of phage-based treatments to deter pathogens such as Aeromonas samonicida, Edwardsiella tarda, and V. harveyi, among others (Richards, 2014 and references therein). Like the vaccination approach, a key feature of phage therapy is its usual pathogen-specific nature, although the extent of specificity of the host-phage interaction may vary in case-dependent manner (Richards, 2014). In this regard, the use of phage mixtures has been considered a reasonable strategy to avoid the development of phage resistance by specific bacterial hosts while enabling the control of diverse pathogens (Richards, 2014). As for the use of vaccines and probiotics, phage dosage and delivery mode (immersion, oral via e.g., live feed ingestion, or injection) are crucial aspects for successful implementation of phage therapy (Richards, 2014; Soliman et al., 2019). To be cost-effective, phase dosage must be the lowest possible to induce bacterial infection with an associated, high phage replication rate (Soliman et al., 2019). Therefore, the use of lytic—instead of lysogenic—phages has been suggested as an imperative for the development of successful phage therapy methodologies (Richards, 2014). Since the ability to isolate and manipulate bacteriophages is strictly limited to the range of culturable bacterial hosts that can be captivated in the laboratory, and because the aquaculture pathobiome may include unculturable, or hard-to-culture, understudied bacteria, an intrinsic hurdle of the phage therapy approach relates with the development of novel methodologies leading to the control of bacterial populations for which no corresponding bacteriophages are known to date. Finally, large-scale application of phage therapy approaches in aquaculture shall be taken with caution, as concerns related with the environmental release of phages and its associated risks have been raised (Meaden and Koskella, 2013). The use of probiotics as a third, microbiome-based therapy approach to disease prevention in aquaculture is addressed below, as well as its likelihood to modulate aquaculture microbiomes toward a sustainable healthy state.

Microorganisms are major contributors to nutrient cycling and functioning within aquaculture facilities yet a fraction of the total microbiome thriving in these man-made is also responsible for disease and mortality affecting live feed, fish larvae, fish, and shellfish. The extent to which solutions to the pathogenicity problem within aquaculture facilities may be found in the naturally occurring aquaculture microbiome is a matter of current scientific debate. The effective implementation of protocols relying on the use of vaccines, phage therapy and probiotics holds promise in deterring pathogen proliferation in intensive fish farming. For instance, a wealth of Gram-positive and Gram-negative bacteria showing remarkable capacities to supress Vibrio pathogens or mitigate vibriosis symptoms in farmed fish and shellfish have been identified in the past 30 years. Nevertheless, to move beyond the proof-of-concept stage, such protocols still face technical and legal challenges that prevent their wide-range applicability at the production scale. Better understanding of microbiomes thriving in “healthy” and “diseased” hosts and aquaculture systems is key to instruct researchers in the pursuit of techniques leading to efficient microbiome manipulation and engineering toward safer rearing systems, eventually decreasing the need of using antibiotics and hazardous chemicals to control bacterial diseases in aquaculture.

The increasing pollution of coastal ecosystems caused by sewage and industrial effluent inputs, including oils, fertilizers, and heavy metals, may also result in a negative physiological response of reared fish, favoring the invasion of bacterial pathogens and parasites in rearing systems exposed to such pollutants. Therefore, proper environmental monitoring and ecosystem conservation are fundamental to prevent bacterial disease incidence in aquaculture. Aquaculture facilities may be in fact “hotspots” for gene transfer, as they contain dense and highly diverse bacterial communities whose structure and taxonomic composition result from the combination of current and past use of antibiotics, probiotics, prebiotics as well as other kinds of treatments or methods. In this context, it is important to discern the coding potential present in the mobile gene pool of Vibrio species and assess the intra- and interspecific transferability of these genes, as this bears implications to our understanding to the roles of Vibrio species as disease-causing agents and of the potential switch from commensal to pathogenic behavior based on processes of gene gain and loss in the Vibrio-associated plasmidome. Indeed, Bruto et al. (2017) revealed that pathogenicity of Vibrio crassostreae toward the cultured oyster species Crassostrea gigas is mediated by the acquisition of a large mobilizable plasmid. It is reasonable to argue that such processes mediate virulence of Vibrio pathogens of fish and may be promoted by high host densities in intensive rearing conditions.

Vibriosis is one of the most important diseases causing high mortality rates in the aquaculture industry. According to the meta-analysis discussed in this review, species such as V. alginolyticus and V. harveyi are among the main responsible for epizootic disease outbreaks causing economic losses in this sector, affecting several fish species, including the production of reared gilthead seabream in the Mediterranean zone. As the frequency of antibiotic- and multidrug-resistance Vibrio spp. is growing, constant surveillance and monitoring of antibiotic resistance and pollution must be assured to avoid the development of multi-resistant strains which may pose threats to both ecosystem and human health. Although certain Vibrio species from diseased farmed gilthead seabream were found to be sensitive to tetracycline, oxytetracycline, chloramphenicol and florfenicol, the development of alternative, cost-effective and sustainable pathogen suppression methods in aquaculture is encouraged from an environmental and a human health standpoint. Particularly worrisome is the current rise of human infections caused by environmental and seafood-associated Vibrio species, apparently influenced by climate-change drivers of microbial community assembly in coastal ecosystems, including intensive seafood farming systems.

The major goal of aquaculture production is supplying food for human consumption. Following several decades of heavy use of antimicrobial drugs and antibiotics to boost intensive fish rearing, current prophylaxis approaches that contribute to a more health-oriented management of aquaculture systems are being increasingly recommended to prevent or suppress epizootic disease outbreaks. They involve the use of less dangerous methods such as vaccines, immunostimulants and probiotics/microbiome therapy. This way, it is believed that healthier food for human consumption may be produced and bacterial resistance to antibiotics may be prevented or alleviated, thus reducing the transfer of acquired antibiotic resistance traits to human pathogens via mobile genetic elements.

GMMS-F, IS-C, and RC conceived and designed the study. GMMS-F performed the literature research, prepared figures, and tables. GMMS-F and RC interpreted the data and wrote the first manuscript draft. All authors read and revised the first manuscript draft and approved the final manuscript.

This study was supported by FCT—Fundação para a Ciência e a Tecnologia, I.P., through the research grants PTDC/MAR/112792/2009 and PTDC/BIA-MIC/31996/2017 and by the European Regional Development Fund (ERDF, Project # 031996, operational code ALG-01-0145-FEDER-031966) through the regional operational programs of Lisbon and Algarve, Portugal. This study was also financed by FCT in the scope of the projects UIDB/04565/2020 and UIDP/04565/2020 of the Research Unit Institute for Bioengineering and Biosciences—iBB, and the project LA/P/0140/2020 of the Associate Laboratory i4HB - Institute for Health and Bioeconomy.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.