# Genetic diversity and virulence gene profiling of Vibrio harveyi in a vibriosis-affected European seabass (*Dicentrarchus labrax*) aquaculture tank

Alix da Fonseca Ferreira, Alice Lehmann, Roxane Roquigny, Thierry Grard, Cédric Le Bris

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## To cite this version:

Alix da Fonseca Ferreira, Alice Lehmann, Roxane Roquigny, Thierry Grard, Cédric Le Bris. Genetic diversity and virulence gene profiling of Vibrio harveyi in a vibriosis-affected European seabass (*Dicentrarchus labrax*) aquaculture tank. Marine Pollution Bulletin, 2025, 212, pp.117553. 10.2139/ssrn.4956372. hal-04889440

HAL Id: hal-04889440

https://ulco.hal.science/hal-04889440v1

Submitted on 15 Jan 2025

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# Genetic diversity and virulence gene profiling of *Vibrio harveyi* in a vibriosis-affected European seabass (*Dicentrarchus labrax*) aquaculture tank

A. Da Fonseca Ferreira, A. Lehmann, T. Grard, R. Roquigny and C. Le Bris

Univ. Littoral Côte d'Opale, UMRt 1158 BioEcoAgro, USC ANSES, INRAE, Univ. Artois, Univ. Lille, Univ. Picardie Jules Verne, Univ.

Liège, Junia, F-62200 Boulogne-sur-Mer, France

## Abstract

Aquaculture is crucial in meeting global seafood demand; however, intensification often leads to bacterial diseases that threaten productivity. *Dicentrarchus labrax*, a key species in European aquaculture, is highly vulnerable to vibriosis, primarily caused by *Vibrio harveyi*. This study investigates genetic profiles associated with vibriosis by analyzing *V. harveyi* strains from a seabass farm during 2022 vibriosis outbreaks. Sampling from biofilm and water environments yielded 946 bacterial isolates, of which 56 were identified as *V. harveyi* using MALDI-TOF MS. ERIC-PCR genotyping revealed four distinct profiles. Despite observing variability in the presence of the 80 tested virulence genes, the overall genetic variation among these profiles was not pronounced. Notably, no single genotypic profile was linked to vibriosis. These findings suggest that the presence of virulence genes alone may not predict disease outbreaks, thus highlighting the need for future research on environmental and transcriptional factors to improve disease control in aquaculture systems.

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## 1 Introduction

Aquaculture is a rapidly expanding industry that plays a crucial role in meeting the global demand for seafood. The European seabass (*Dicentrarchus labrax*) is among the most economically significant species in European aquaculture. Its production has experienced substantial growth, reaching 80,786 metric tons and a value of 470 million euros in 2020 in Europe (EUMOFA, 2022). Despite this growth and progress in husbandry techniques, the European seabass remains highly susceptible to diseases such as vibriosis, caused by bacteria of the *Vibrio* genus. Vibriosis can lead to significant mortality events, posing a major challenge to the industry, with symptoms such as ulcers, hemorrhages, and tissue necrosis. Among the Harveyi clade, *Vibrio harveyi* is particularly problematic in European seabass aquaculture due to its pathogenicity and virulence (Zhang et al., 2020). A critical early step in studying *V. harveyi* is differentiating it from other species within the clade, as their genetic similarities make accurate identification difficult. Furthermore, not all *V. harveyi* strains necessarily provoke vibriosis. Methods like ERIC-PCR (enterobacterial repetitive intergenic consensus-polymerase chain reaction) have been effectively used to investigate genetic variation among *V. harveyi* isolates, which can help to explain differences in virulence and pathogenicity (Bai et al., 2020; Xu et al., 2017). In a pivotal study, Xu et al. (2017) applied ERIC-PCR to *V. harveyi* strains isolated from cultured fish in southern China, demonstrating its effectiveness in distinguishing between pathogenic and non-pathogenic strains so as to create genetic fingerprints linked to virulence and identify specific isolates associated with mortality events.

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Building on these findings, it is essential to further investigate how different genotypic profiles correlate with variations in virulence. This is particularly important since the pathogenic mechanisms of *V. harveyi* are complex and not yet fully understood, in part due to the species' high genetic diversity. Thus, it is necessary to focus on known virulence genes in *V. harveyi*, in the literature and databases, as well as potential virulence genes in other *Vibrio* species. These bacteria are prone to gene transfer both within and across species, a phenomenon that is facilitated by their ability to form biofilms (Fu et al., 2022). In aquaculture systems, bacterial communities exist as biofilms on surfaces and as planktonic bacteria in the water column. The virulence of these two forms may differ, highlighting the need to study both kinds of isolates in order to manage disease outbreaks effectively. In this study, we collaborated with a farm that experienced two vibriosis outbreaks in July and August 2022. It was crucial to analyze bacterial isolates from these episodes to gain insights into the strains potentially involved. Sampling was conducted to identify the strain(s) responsible. Initially, *Vibrionaceae* were collected, and bacterial isolates were identified by MALDI-TOF MS and a combination of the Bruker and Luvibase databases (Mougin et al., 2020). Following identification, ERIC-PCR was used to type the *V. harveyi* isolates and categorize them into genomic profiles. To provide insights into *V. harveyi* pathogenesis, the presence in isolates of 80 virulence genes potentially implicated in vibriosis was subsequently investigated. By elucidating the genetic diversity and virulence characteristics of *V. harveyi* in both biofilms and planktonic phases, the goal was to try to link a genetic profile with vibriosis outbreaks, and to identify the major virulence genes associated with this profile for future transcriptomic studies. This approach could be applied to detect the emergence in aquaculture tanks of problematic strains early on, thereby facilitating the development of targeted antivirulence strategies against these major virulence genes. Ultimately, this would help advance more sustainable aquaculture practices.

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# 2 Materials and methods

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## 2.1 Sample collection and treatment

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Between May and November 2022, we conducted a sampling campaign at Aquanord-Ichtus, an aquaculture facility in Gravelines (France), along the southwest coast of the North Sea (Da Fonseca Ferreira et al., 2024). The chosen outdoor aquaculture tank for the study had a volume of approximately 975 cubic meters and accommodated up to 68,000 sea bass, with an average density of 32 kg per cubic meter. Daily feeding and oxygenation were maintained to ensure optimal dissolved oxygen levels. During the study period, two outbreaks of vibriosis attributed to *Vibrio harveyi* were recorded by a veterinary laboratory collaborating closely with the farm (data not shown). These outbreaks occurred in July and August, necessitating two rounds of curative oxytetracycline treatment exclusively prescribed by the veterinarian. Sampling followed the methodology described in Da Fonseca Ferreira et al. (2024). Briefly, biofilm samples were collected using five cylindrical concrete specimens positioned at three depths (0.3 m, 2.15 m, and 4 m) on different ropes along the tank walls. Additionally, 15-liter water samples were collected per sampling, with five 1-liter samples taken
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at each depth using a submersible pump. Sixteen sampling events were conducted, resulting in a total of 240 individual water samples and 240 biofilm samples over the study period. Water samples were filtered through 0.45-µm and 0.22-µm nitrocellulose filters to capture bacteria. Concrete specimens were retrieved, preserved in sterile water, and swabbed. Samples were stored in Luria-Bertani broth with 20% NaCl and glycerol at -80 °C. Prior to analysis, samples were thawed, vortexed, and centrifuged to obtain cell pellets. A 100-µL aliquot from each sample was enriched in 1 mL of LBS for 24 hours, followed by inoculation onto thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Becton Dickinson Difco, Franklin Lakes, NJ, USA) using a 10 µL inoculation loop. All agar plates were then incubated overnight at 35 °C, which allows growth for many Vibrio species (Farmer III et al., 2015). One colony of each visually different morphotype was randomly selected from each plate and further individually cultured on marine agar (MA; Condalab, Madrid, Spain) at 35 °C overnight. Pure bacterial cultures were preserved in LBS broth supplemented with 20% glycerol and stored at -80 °C until identification through MALDI-TOF MS.

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## 2.2 Bacterial identification using MALDI-TOF MS

After thawing, 10 µL of bacterial isolates was cultured and incubated for 24 hours at 35 °C in 2 mL of marine broth (MB) (Becton Dickinson Difco, Franklin Lakes, NJ, USA) in a 96 deep-well Masterblock (Greiner Bio-One, Frickenhausen, Germany). The isolates were identified using MALDI-TOF by the EQUIPEX REALCAT platform (Lille, France). Briefly, the fresh bacterial cultures were centrifuged, and the pellets extracted using 70% formic acid (Alfa Aesar, Kandel, Germany) and 100% acetonitrile (Biosolve Chimie). Protein extracts were then deposited in four replicates on a ground steel MTP 384 target plate (Bruker Daltonics, Bremen, Germany) overlaid with an α-cyano-4-hydroxycinnamic acid matrix (Sigma-Aldrich, St. Louis, MO, USA), and analyzed using the Autoflex Speed MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). MALDI-TOF MS profiles were compared with profiles in the Bruker BioTyper database (version 9.0.0.0) implemented with an in-house Vibrio database known as Luvibase (Mougin et al., 2020). The level of concordance between the experimental MALDI-TOF MS profiles and the MSPs was quantified using a log score. Specifically, a log score greater than 2.3 indicated a highly probable identification at the species level, while a lower log score ranging from 2.0 to 2.3 indicated a probable identification. Scores within the range of 1.7 to 2.0 suggested a probable identification at the genus level, while scores below 1.7 indicated a notable absence of substantial similarities between the MSPs contained in the databases and the unknown sample profile. Identification was considered reliable with scores of at least 2 for 3 out of 4 deposition replicates.

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## 2.3 ERIC-PCR and GELJ analysis

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All the isolates identified as V. harveyi by MALDI-TOF-MS were grown overnight at 35 °C on TCBS plates. DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations. The purity of the DNA was assessed using a NanoDrop 8000 spectrophotometer (Thermo
Scientific, Waltham, U.S.), and the concentration of samples was measured with the Qubit™ dsDNA Broad Range Assay Kit on a Qubit™ 4 fluorometer (Thermo Scientific, Waltham, U.S.). ERIC-PCR was conducted in a final volume of 25 µL, containing 12.5 µL of Master Mix PCR (2x) (Thermo Fisher Scientific, Waltham, MA, USA), 8 µL of sterile water, 1 µL of each ERIC primer at 10 pmol/µL (ERIC1R: 5'-ATGTAAGCTCCTGGGGATTCAC-3' and ERIC2: 5'-AGTAAGTGACTGGGGTGAGCG-3'; TIB MOLBIOL, Syntheselabor GmbH, Germany) (Xu et al., 2017), and 2.5 µL of extracted DNA (20 ng/µL). The amplification step was performed using a Bio-Rad iCycler (Bio-Rad, Hercules, CA, USA) with the following cycling conditions: initial denaturation at 95 °C for 7 min, followed by 35 cycles of denaturation at 90 °C for 30 seconds, annealing at 52 °C for 1 min, then elongation at 65 °C for 8 min. A final elongation step was carried out at 68 °C for 16 min. Subsequently, 10 µL of a mixture of 10 µL of PCR products and 3 µL of loading buffer (6X) (Lonza, Basel, Switzerland) were loaded onto a 1.5% agarose gel and stained with ethidium bromide (1% solution) (Fisher BioReagent, Powai, India). Electrophoresis was performed using a FastGene 100 bp DNA marker (Nippon Genetics, Tokyo, Japan) as a reference. DNA was migrated on gel at 120 V for 60 min. UV transillumination was used for visualizing results, and images were captured using an E-Box CX5 TS Edge (Vilber, Marne-la-Vallée, France). Each ERIC-PCR assay was performed in at least triplicate. The ERIC-PCR fingerprints of amplified DNA fragments, obtained through agarose gel electrophoresis, were analyzed using GelJ software (version 2.3), designed for constructing phylogenetic trees from DNA fingerprints. Band positions were normalized using the FastGene 100 bp DNA marker (Nippon Genetics), with manual correction for accuracy. A dendrogram was generated to represent the genetic relatedness among *Vibrio harveyi* isolates. The Dice similarity coefficient and UPGMA method were used to assess and cluster the profiles, resulting in the classification of the *V. harveyi* isolates into four distinct profiles with an 83% similarity cut-off.

## 2.4 Primer design for virulence genes and PCR

### 2.4.1 Virulence gene selection and primer design

From the sequenced genomes of seven vaccine strains recovered from the aquaculture farm (data neither shown nor published), a predictive list of virulence genes was established by GenoScreen (Lille, France) for each isolate. We further analyzed these genomes using VF Analyzer (based on the Virulence Factor Database, VFDB) to identify virulence genes in each genome (consulted on September 2022). FASTA sequences were analyzed with VF Analyzer to confirm the consistency of virulence gene identification by GenoScreen. Seventy-six genes common to at least two vaccine strains were thus selected for primer design. To complement this list, four genes identified through the literature and not listed among the 76 genes previously mentioned were included: *V. harveyi* R Toxin (*toxRvh*), *V. harveyi* hemolysin (*vhh*), thermostable direct hemolysin (*tdh*), and a gene involved in the flagellar system (*flaC*) (Table S1). Virulence gene sequences were retrieved from the vaccine strains' genomes using the CLC Genomics Workbench (QIAGEN, version 12.0.1). Sequences of the genes added from the literature were taken from the NCBI with the following accession numbers: DQ503438.1
(toxRvh), JN033877.1 (vhh), NZ_QOUW02000047.1 (tdh) and EU240947.1 (flaC). The 80 primer pairs were designed using Primer3Plus (version 3.2.6), avoiding potential primer dimers or hairpin structures when possible. If unavoidable, primers with secondary structures having a ΔG (Gibbs free energy change) greater than -3.0 kcal/mol were selected. All primers were validated using the CLC Genomics Workbench with seven vaccinal genomes and BLAST to ensure specificity to V. harveyi and a lack of mutations. All primers had a melting temperature (Tm) around or equal to 60 °C and a GC content close or equal to 55%. Primers were aligned on the genomes of the vaccine strains to ensure that they were present in a single copy. The primers (Table S1) were ordered from TIB MOLBIOL (Syntheselabor GmbH).

### 2.4.2 PCR for virulence genes

To explore a potential correlation between the occurrence of virulence genes in V. harveyi and their ERIC profiles while trying to limit the number of isolates to study, two isolates maximum per profile were selected for complementary analysis, except for profile 2 which included only one isolate, resulting in a total of seven isolates being profiled. The 80 virulence genes were screened using PCR across these seven V. harveyi selected isolates. A no-template control was included and each virulence gene PCR was conducted at least in duplicate. To evaluate the PCR functionality of virulence genes, the 80 primer pairs were tested on the isolate they were designed from. Each primer pair underwent triplicate testing alongside a no-template control to verify its functionality. Each PCR reaction was conducted in a 25 µL volume, consisting of 12.5 µL of 2x Master Mix PCR (Thermo Fisher Scientific), 9 µL of sterile water, 0.5 µL of each primer at 10 pmol/µL, and 2.5 µL of extracted DNA (20 ng/µL). The reactions were carried out using a Bio-Rad iCycler (Bio-Rad) with a standardized protocol for all genes: initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 45 s, concluding with a final extension step at 72 °C for 10 min. Subsequently, 10 µL of a mixture of 10 µL PCR products and 3 µL loading buffer (6X) (Lonza) were deposited on 1.5% agarose gels stained with ethidium bromide (1% solution) (Fisher BioReagent). Electrophoresis was performed in the same way as above with ERIC-PCR.

## 3 Results

### 3.1 Identification of isolates

A total of 946 bacterial isolates—383 from biofilm and 563 from water—were collected using TCBS agar (Figure 1). They were all identified with a MALDI-TOF analysis using Bruker and Luvibase databases, and identification was reliable, with scores of at least two for three out of four replicates. Among the biofilm samples, 309 isolates were identified as *Vibrio alginolyticus*, while 482 isolates were found in the water samples. *Vibrio harveyi* was also identified in 31 biofilm isolates and 25 water isolates. Other notable species included *Vibrio parahaemolyticus*, with 29 isolates in total (11 in biofilm samples and 18 in water samples), *Vibrio rotiferianus* with seven isolates (four in biofilm samples and three in water samples), and *Vibrio owensii* with five isolates (three in biofilm samples and two in water samples). Additionally, certain species were unique to the water
samples: *Shewanella algae* with seven isolates, *Vibrio mytili* with two isolates, *Vibrio diazotrophicus* with one isolate, and *Shewanella indica* with one isolate.

## 3.2 *V. harveyi* ERIC-PCR fingerprinting

The 56 isolates of *Vibrio harveyi* were thoroughly examined using ERIC-PCR profiling, revealing significant genetic diversity among the isolates. The similarity indices of the dendrogram constructed from the ERIC-PCR profiles ranged from 75% to 100%, indicating a broad spectrum of genetic diversity (Figure S1). Using an 83% cut-off, the isolates were classified into four distinct ERIC-PCR genotypes. This classification facilitated the analysis of genomic profile distribution according to the sampling month, type of sample (water or biofilm), and sampling depth (top, middle, bottom) (Figure 2). The analysis of the distribution of *V. harveyi* profiles across the water column and months revealed a consistent presence of the species from May to November, with varying ERIC profile distributions (Figure 2). Profile 1 contained four isolates, primarily collected from the bottom of the tank in November. Profile 2 was represented by a single isolate collected in July from the middle of the tank, coinciding with the first observed outbreak of vibriosis. Profile 3 comprised 41 isolates and was consistently found throughout the sampling period across all depths (top, middle, bottom) and in both water and biofilm samples. Profile 4 contained 10 isolates, predominantly collected from biofilms (eight out of 10). This profile emerged in July, persisted throughout August during the second vibriosis outbreak, reappeared in October, and disappeared in November.

## 3.3 Genotyping of virulence genes in the profiles

After successfully validating the designed primers and confirming their amplicon sizes, we investigated the occurrence of virulence genes among two isolates per distinct *V. harveyi* genomic profile (Figure 3). The isolates representing each profile were as follows: profile 1 included isolates B15B3-3 and E15B5-1; profile 2 consisted of isolate E4M2-2 (only isolate); profile 3 included isolates E1M3-3 and B4M5-1; and profile 4 included isolates B10B5-2 and B13M1-1 (internal nomenclature). Among the seven isolates studied, a majority harbored most of the genes examined, but several significant differences were noted. Profile 1 contained all the screened genes associated with chemotaxis signaling, flagellar assembly and function, pilus assembly, secretion system, hemolysin, iron acquisition, exopolysaccharide (EPS) biosynthesis, capsule, and toxin regulation. These isolates shared nearly identical sets of genes crucial for environmental adaptation and host interaction, with minor variations observed in E15B5-1, which harbored multiple bands for genes *flaB*, *motX*, *motY*, *mshG*, and *mshL*. Profile 2, characterized by isolate E4M2-2, lacked the *mshA* gene but had all the other analyzed genes. Profile 3 contained all the genes except for the virulence-associated *vcrV* in E1M3-3 and lacked *mshA* in B4M5-1, which is crucial for pilus biosynthesis. Profile 4, comprising isolates B10B5-2 and B13M1-1 isolated from biofilms, showcased distinct genetic profiles with specific gene deficiencies. B10B5-2 lacked flagellar genes *flgD*, *flgF*, *flgK*, *flgL*, *flhF*, *fliK*, *fliR*, *flrB*, pilus gene *pilB*, virulence factors *vcrV*, *vhh*, *vctA*, and EPS biosynthesis gene *epsF*. In contrast, B13M1-1 lacked flagellar genes *flgD*, *flgE*, *flgG*, *flgH*, *flgM*, *flhA*, pilus
assembly gene mshA, and EPS biosynthesis gene epsF, and possessed multiple bands for mshG, mshL, and vcrD genes, along with a shorter variant of vscC. This indicates that isolates from the same profile do not systematically share exactly the same genes.

## 4 Discussion

The mere presence of *Vibrio harveyi* in aquaculture environments does not necessarily lead to vibriosis outbreaks (Mougin et al., 2021), as the development of the disease is influenced by a complex interplay of abiotic and biotic factors (Montánchez and Kaberdin, 2020). Although *V. harveyi* is a well-established primary causative agent of vibriosis, its virulence potential is also shaped by what virulence genes the isolates in the tank harbor. The overarching goal of our study was to identify the genetic profile(s) linked to vibriosis and associated mortality. Our study specifically aimed to investigate the genetic diversity of *V. harveyi* in both water and biofilms, characterize its virulence gene profiles, and analyze its relative temporal and spatial distribution on a sea bass farm in northern France. The ultimate goal is to develop a method for detecting problematic bacteria in the tanks before they cause disease outbreaks. Sampling was conducted from May to November 2022. During this period, the farm experienced two significant vibriosis outbreaks attributed to *V. harveyi*, one in July and another one in August. The unpredictable nature of the *Vibrio harveyi*-associated vibriosis episodes in the farm further highlights the need to explore the genetic diversity of the strains and attempt to understand which ones are responsible for these outbreaks. In analyzing the bacterial populations within the tank, we used TCBS selective media to isolate a total of 946 visually distinct colonies from both biofilm (383 isolates) and water samples (563 isolates), with a focus on *Vibrionaceae* but also including some other species. MALDI-TOF identification revealed a variety of *Vibrio* species, with *Vibrio alginolyticus* being the most prevalent. This species accounted for 309 isolates from biofilm and 482 from water samples. *Vibrio alginolyticus* thrives in warm coastal waters and is often associated with severe infections and high mortality in species like sea bass, making it a significant concern in aquaculture (Abdel-Aziz et al., 2013). *Vibrio alginolyticus* is not the sole problematic species in our case, as *V. harveyi*, the causative agent of two outbreaks during the campaign, was also present, though in smaller numbers (31 biofilm isolates and 25 water isolates). Furthermore, the bacterial isolates were selected visually, which may have led to more *V. alginolyticus* colonies being isolated due to greater morphological variation, while *V. harveyi* might have had more visually similar isolates. In a previous study conducted at the same farm in 2018, when no vibriosis outbreaks occurred and sampling was limited to the surface water of a smaller tank (75 m³, 1.3 meters deep), *V. harveyi* and *V. alginolyticus* were present in approximately equal amounts, along with *Photobacterium damselae* subsp. *damselae* regardless of the sampling site and sample type (Mougin et al., 2021). This earlier finding underscores the need to consider both biotic and abiotic factors to better understand vibriosis development. Under favorable environmental conditions, co-infection with *V. harveyi* can significantly enhance the pathogenic effects of other species. For instance, co-infection between *V. harveyi* and *V. alginolyticus* has been
shown to worsen vibriosis symptoms in hybrid groupers (*Epinephelus polyphekadion* × *E. fuscoguttatus*) (Mohamad et al., 2019). Studies also indicate that *V. harveyi* and *V. alginolyticus* were also found with *V. parahaemolyticus* in superficial lesions among Asian seabass (*Lates calcarifer*) (Liu et al., 2016). Several bacteria other than *Vibrio* species were identified in our samples, including *Shewanella* algae and *Shewanella indica*, which were unique to biofilm samples. *Shewanella* species have been implicated in mixed infections with *Vibrio* species across various teleost fish species, further underscoring the increased threat of mixed infections in aquaculture (Sony et al., 2021). In light of the challenges in examining all the concurrent species during *Vibrio* outbreaks, it is essential to concentrate on *V. harveyi* to determine whether some genetic profiles are specific to vibriosis episodes. To classify and group genetically similar *V. harveyi* isolates present during the campaign, we employed ERIC-PCR, which generates detailed 'fingerprint-like' profiles for each isolate. This technique is cost-effective and well-suited for analyzing genetic diversity in *Vibrio* species, including *V. harveyi* (Bai et al., 2020; Kahla-Nakbi et al., 2006; Su and Chen, 2020; Xu et al., 2017; Yang et al., 2021). In our study, ERIC-PCR revealed distinct banding patterns among the isolates, with amplicon numbers ranging from 4 to 11 and fragment sizes from 100 bp to over 4 kb, underscoring the substantial genetic diversity within the *V. harveyi* population. The 56 isolates were classified into four ERIC-PCR genotypes (Figure S1) using an 83% similarity cut-off. However, this classification does not produce homogeneous groups in terms of number of isolates, leading to uneven distribution of the isolates across the profiles. For each profile, isolates were selected to represent variation in one parameter: type (biofilm or water), depth (top, middle, bottom). To evaluate if any profile exhibited increased pathogenic potential, we screened 80 genes associated with virulence in the seven isolates representing the diversity of genetic profiles (Figure 3). Within these profiles, we observed differences in the occurrence of genes. In profile 1, one isolate out of two, originating from the bottom of the water column, showed multiple amplicons for motility and pilus assembly genes. This phenomenon may be attributed to the presence of multiple gene variants, where imperfect hybridization during amplification forms heteroduplexes that migrate more slowly during electrophoresis than perfectly matched pairs, resulting in multiple bands (Moreno et al., 2002). Profile 2 included a single isolate, exhibiting all the studied genes except *mshA*. The absence of *mshA* reduces the ability to form biofilm, crucial for environmental persistence, and increases resistance to VGJ4 phage infection (Ledón et al., 2012; Sinha-Ray and Ali, 2017). Therefore, this isolate is particularly intriguing and merits further study due to its possession of several virulence genes, potential filamentous bacteriophage resistance, and its presence during the initial vibriosis outbreak month. Compared with other profiles with multiple isolates, it would be valuable to obtain additional isolates with this profile to confirm the presence or absence of these genes. Profile 3 was the most sampled, potentially because it encompasses the majority of isolates—41 in total—collected from various depths (top, middle, and bottom) as well as from both water and biofilm samples. The large number of isolates in this profile may explain the genetic differences observed between the two isolates representing it.
Therefore, further investigation of additional isolates using ERIC-PCR would be beneficial, as analyzing only two isolates may not fully capture the genetic diversity within this profile. Profile 4 included 10 isolates, predominantly collected from biofilm samples. Both of the profile 4 isolates selected exhibited distinct genetic adaptations compared with the other three profiles, highlighting specialized strategies for biofilm survival. Both isolates displayed deficiencies in flagellar genes, potentially reducing motility, favoring attachment within biofilms, and decreasing virulence (McGee et al., 1996). Like profile 3 isolates, profile 4 isolates showed absences or multiple bands for flagellar, pilus assembly and secretion system genes, though they were not the same in the two isolates. They also exhibited deficiencies in *epsF*, suggesting reduced EPS biosynthesis (Kong et al., 2022). One isolate in profile 4 also lacked *vhh* and *vctA*, genes associated with hemolysis and iron acquisition, respectively, which could potentially impair their pathogenicity. The other isolate had a truncated amplicon for the *vscC* gene, possibly due to a deletion that could impair its function and render *V. harveyi* less virulent and more susceptible to abiotic factors (Mao et al., 2021; Zhang et al., 2021). Given the observed diversity both among and within profiles, it would be useful to investigate whether there is a temporal pattern or succession of profiles that could explain the development of vibriosis. Analyzing the temporal distribution of *V. harveyi* profiles across the water column revealed that the species was consistently present from May to November, with variations in isolate and ERIC profile distributions (Figure 2). This finding is supported by qPCR quantification, which confirmed the continuous presence of *V. harveyi* throughout the sampling campaign (Da Fonseca Ferreira et al., 2024). Profile 1 was primarily collected in November. Profile 2, collected in July, coincided with the first vibriosis outbreak. Profile 3 was consistently found throughout the sampling period. Profile 4, predominantly collected from biofilms, emerged in July, persisted through the second outbreak in August, reappeared in October, and disappeared in November. However, when examining the virulence genes, it is not clear whether a specific profile is responsible for the vibriosis outbreaks, as nearly all the profiles contain most of the targeted genes. The only exception is profile 4, which diverges from the others by lacking some key genes. Like Xu et al. (2017), our study identified genetic diversity among *V. harveyi* isolates, but a major difference is that they first conducted pathogenicity challenges, grouping all pathogenic strains into the same profile. Within this profile, not all strains possessed the virulence genes they investigated. In contrast, our study focused on environmental isolates and aimed to detect genetic diversity linked to the timing and occurrence of vibriosis outbreaks, resulting in the identification of four distinct profiles, most of which contained virulence genes. However, given the large number of virulence genes involved in various mechanisms, it is challenging to determine whether a specific profile is responsible for the development of vibriosis. While ERIC-PCR proved to be a rapid, efficient, and cost-effective method for distinguishing isolates, it did not pinpoint a specific profile linked to vibriosis. Our findings suggest that merely detecting virulence genes may not fully capture the pathogenic potential. However, this work is crucial for understanding genetic diversity and guiding future research, which should focus on exploring these virulence factors under varying
309 environmental and host-specific conditions to mimic natural vibriosis outbreaks. This includes conducting 310 bacterial challenge studies to test the isolates, to identify which ones are pathogenic and which ones are not. 311 Subsequently, it is essential to consider the expression of the studied virulence genes in those that cause 312 disease, as recommended in studies of Vibrio fish infections (Hernández-Cabanyero et al., 2022; Wang et al., 313 2022). By unraveling the transcriptional dynamics of these virulence genes, future studies could advance our 314 understanding of how V. harveyi induces vibriosis in D. labrax. Such investigations into genetic diversity, 315 virulence genes, and pathogenic mechanisms are pivotal for predicting disease outbreaks and developing 316 targeted strategies for disease prevention and control in aquaculture settings. 317

## 5 Conclusion 317

318 This study highlights the complex dynamics of Vibrio harveyi in aquaculture, focusing on its genetic diversity 319 and virulence potential. Our findings reveal two key aspects of V. harveyi's role in vibriosis outbreaks on a sea 320 bass farm. First, ERIC-PCR revealed genetic diversity among V. harveyi isolates, identifying distinct genotypic 321 profiles. This variability in gene occurrence may influence the pathogen's behavior and adaptability, indicating 322 that V. harveyi isolates are not uniform. Such diversity could enhance the pathogen's persistence and its 323 interactions with the environment and host. Second, the analysis of virulence genes across profiles revealed 324 that while many isolates possess genes linked to pathogenicity—such as those for biofilm formation, motility, 325 and EPS biosynthesis —no single profile was clearly and specifically associated with vibriosis outbreaks. This 326 suggests that virulence potential is influenced more by the interplay between genetic diversity and 327 environmental factors than by the presence of individual genes alone. These findings emphasize the critical 328 need to consider both biotic and abiotic environmental factors along with strain-specific virulence profiles 329 when evaluating the risk and management of vibriosis in aquaculture systems (Da Fonseca Ferreira et al., 2024; 330 Mougin et al., 2021; Roquigny et al., 2021). Future research should explore how genetic diversity impacts 331 virulence under various environmental and host conditions, and investigate the transcriptional responses of 332 virulence genes. Given the complexity of the tripartite interaction, one goal is to understand the influence and 333 relationship between each part to better anticipate vibriosis outbreaks. These studies are crucial for enhancing 334 predictions and management of disease outbreaks in aquaculture systems, as global warming is likely to alter 335 the distribution and abundance of Vibrio species, and increase the frequency and severity of such events (Ma 336 et al., 2023; Trinanes and Martinez-Urtaza, 2021). 337

## 6 Acknowledgements 337

338 The authors would like to thank Aquanord-Ichtus, and especially Guillaume Tielie, Annabelle Duhamel, and 339 Céline Doyen. We are also grateful to the REALCAT platform funded by a French government grant 340 administered by the French National Research Agency (ANR) as part of the "Investments for the Future" 341 program (ANR-11-EQPX-0037). We would also like to thank Enola Blaise, Lorédane Clause and Margaux Lay
342 for technical support, and Dr Christophe FLAHAUT for his expertise in mass spectrometry. Alix Da Fonseca
343 Ferreira would like to thank the PMCO council and Hauts-de-France regional council for their financial support
344 of her PhD studies. This work was funded by the French government, Ifremer and the Hauts-de-France region
345 in the framework of the CPER 2021-2027 IDEAL project and the IFSEA Graduate School (stemming from the
346 National Research Agency under the "Investments for the Future" program, ANR-21- EXES-00 11).

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Preprint not peer reviewed.
a.

Vibrio alginolyticus
309

Total
383

Vibrio owensii (3)
Vibrio rotiferianus (4)

Vibrio parahaemolyticus
11

Unidentified

25

Vibrio harveyi
31

31

b.

Vibrio alginolyticus
482

Total
563

Vibrio parahaemolyticus
18

Unidentified

Vibrio harveyi
22

25

Shewanella indica
1

Vibrio diazotrophica
1

Vibrio mytili
2

Vibrio owensii
2

Vibrio rotiferianus
3

Shewanella algae
7
<figure><img src="image_3.png" /></figure>
<table><thead><tr><th colspan="2">PROFILE 1</th><th colspan="2">PROFILE 2</th><th colspan="2">PROFILE 3</th><th colspan="2">PROFILE 4</th><th></th></tr><tr><th>B15B3-3</th><th>E15B5-1</th><th>E4M2-2</th><th></th><th>E1M3-3</th><th>B4M5-1</th><th>B10B5-2</th><th>B13M1-1</th><th></th></tr></thead><tbody><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>cheA</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>cheB</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>cheR</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>cheV</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>cheW</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>cheY</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>cheZ</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flaA</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flaB</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flaC</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flaE</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flaG</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flaI</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flgB</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flgC</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flgD</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flgE</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flgF</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flgG</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flgH</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flgK</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flgL</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flgM</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flhA</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flhB</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flhF</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flhG</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmA</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmD</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmE</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmF</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmG</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmH</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmI</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmJ</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmK</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmL</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmM</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmP</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmQ</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmR</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmS</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmA</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmB</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>flmC</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>motA</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>motB</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>motX</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>motY</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>mshA</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>mshE</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>mshF</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>mshG</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>mshH</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>mshI</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>mshL</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>pldB</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>gspD</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>vcrD</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>vcrH</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>vcrV</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>vscC</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>vscl</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>vsclL</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>vsclN</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>vxsC</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>thh</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>tdh</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>vhh</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>hutA</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>vctA</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>epsC</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>epsE</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>epsF</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>exopolysaccharide biosynthesis</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>epsK</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>epsL</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>cpsA</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>cpsH</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>toxRvh</td></tr></tbody></table>

Chemotaxis

Flagellar assembly & function

Pilus assembly

Secretion system

Hemolysin

Iron acquisition

Exopolysaccharide biosynthesis

Capsule

Toxin regulation

Gene occurrence :

Present

Absent

Multiple bands

Smaller band

Research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=234200
## Figures captions

Figure 1: Donut plots of species found in the bacterial isolates sampled in an aquaculture tank, grown on TCBS agar and identified using MALDI-TOF mass spectrometry. Plot a. shows bacteria collected in the biofilm samples, and b. shows bacteria collected in the water samples.

Figure 2: Distribution of the ERIC profiles of the 56 *Vibrio harveyi* isolates and their cumulative appearance over months, sample type, and depth (T: top; M: middle; B: bottom) collected during sampling in the aquaculture tanks. The months with recorded vibriosis outbreaks are highlighted in green.

Figure 3: Heatmap of the potential virulence genes studied across the four identified ERIC profiles and their functions. Green: the gene is present with an expected amplicon size; red: the gene is absent; blue: the expected amplicon is present but with multiple bands; yellow: the amplicon is smaller than expected.