non-*V. harveyi* isolates, and compared with two pairs of primers targeting the *topA* and *toxR* genes that were designed previously. All sets of primers were able to distinguish *V. harveyi* from closely related species belonging to the Harveyi clade. However, the *mreB* primers showed better inclusivity and were thus used to develop the real-time PCR assay. A quantification curve was obtained from pure culture of *V. harveyi* and exhibited excellent efficacy with detection levels as low as 5 genome copies in the PCR reaction. After selection of the extraction kit allowing the best DNA quantity and purity, validation was performed on both seawater and biofilm samples collected from a fish farm. The presence of inhibitors in the DNA templates was evaluated and a 10-fold dilution of template DNA was recommended in order to avoid their effects. The assay was able to detect *V. harveyi* from environmental samples, confirming the validity of the method. This real-time PCR method will help to evaluate the dynamics of *V. harveyi* in aquaculture facilities. Suitable prophylactic control measures could be designed using this method, instead of the use of curative methods such as antibiotics.

# 1. Introduction

*Vibrio* are Gram-negative bacteria ubiquitous in aquatic environments (Farmer *et al.*, 2005). Some of them, such as *Vibrio harveyi*, are known to be the causative agents of vibriosis, a fatal hemorrhagic septicemia disease affecting aquatic animals (Austin *et Zhang*, 2006). Vibriosis caused by *V. harveyi* is a major concern in aquaculture. More particularly, this species is a well-known pathogen of shrimps, mollusks and crustaceans (Sawabe *et al.*, 2007b, Travers *et al.*, 2008, Soto-Rodriguez *et al.*, 2012). Infections have also been reported in fishes such as seabass, *Dicentrarchus labrax* (Pujalte *et al.*, 2003). Recently, Vendramin *et al.* (2016) identified *V. harveyi* as an emerging problem in reared seabass, an economically important fish species in Europe (FAO 2005-2020). Importantly, Bourne *et al.* (2006) highlighted that biofilms on rearing tanks may act as potential reservoirs for *V. harveyi*. Biofilms of *V. harveyi* are even more dangerous to reared animals given that they can enhance the resistance of bacteria to antibiotics or water sanitizers (Karunasagar *et al.*, 1996, Thompson *et al.*, 2004a). This phenomenon leads
to persistence of *V. harveyi* in aquaculture tanks. Early diagnosis and monitoring of *V. harveyi*
prevalence are therefore major concerns in seabass farming systems.

Conventional culture-based and biochemical methods for the detection and identification of
*V. harveyi* are unreliable, laborious, and time-consuming (Bonnin-Jusserand *et al.*, 2017). On the
contrary, polymerase chain reaction (PCR)-based methods offer a rapid, reliable, and more
specific analysis (Cano-Gomez *et al.*, 2009). A number of conventional PCR methods have
already been reported for the detection of *V. harveyi* in farming systems (Conejero et Hedreyda,
2003, Oakey *et al.*, 2003, Pang *et al.*, 2006, Kim *et al.*, 2014b). However, to our knowledge, only
two studies have described real-time PCR methods for the quantification of *V. harveyi* in
seawater coming from an abalone farm (Fukui et Sawabe, 2008, Schikorski *et al.*, 2013). These
studies did not process complex samples such as biofilms, and did not include a wide range of
environmental isolates or several closely *V. harveyi*-related species belonging to the Harveyi
clade. They also stated that improvements in their protocols remain to be made in order to
increase the sensitivity of their methods. Furthermore, Fukui et Sawabe (2008) highlighted the
lack of specificity of their designed oligonucleotides, leading to an unreliable estimation of the
amount of *V. harveyi* in seawater. Identification of reliable target genes of *V. harveyi* remains
difficult due to genome similarity between species belonging to the Harveyi clade (Gomez-Gil *et
al.*, 2004). The standard target 16S rRNA gene, commonly used, is not suitable to distinguish
these species from one another. For instance, *V. harveyi*, *V. campbellii*, and *V. rotiferianus* share
more than 99% sequence similarity in the 16S rRNA gene (Gomez-Gil *et al.*, 2003, Gomez-Gil *et
al.*, 2004). Cano-Gomez *et al.* (2011) performed a multilocus sequence analysis (MLSA) based on
the 16S rRNA, *mreB*, *ftsZ*, *pyrH*, *rpoA* and *topA* genes, and results showed that the
concatenation of both the *mreB* and *topA* loci was sufficiently accurate to differentiate between
*V. harveyi*-related species. Therefore, using only the *mreB* (rod shape-determining gene, subunit
B) or *topA* (topoisomerase I) gene could potentially enable discrimination by PCR. In addition,
Pascual *et al.* (2010) performed an MLSA study based on the 16S rRNA, *gyrB*, *pyrH*, *rctB*, *recA*,
*rpoD* and *toxR* genes. They suggested that the individual *toxR* (transmembrane transcriptor
regulator) gene has high discriminatory power and could be suitable to differentiate between
*V. harveyi*-related species. Oligonucleotides targeting the *topA* and *toxR* genes have already been designed (Pang *et al.*, 2006, Cano-Gomez *et al.*, 2015). Their specificity still needs to be clarified since the studies did not include a wide range of environmental isolates or several closely *V. harveyi*-related species. However, no oligonucleotide targeting the *mreB* gene of *V. harveyi* has been described so far.

This study reports the development of a specific SYBR Green I real-time PCR method for the detection and quantification of *V. harveyi* in both seawater and biofilm samples from aquaculture tanks. The optimized method provides a common protocol to extract *V. harveyi* DNA from both of these complex environmental samples, while minimizing the impact of PCR inhibitors. The specificity of the previous *topA* and *toxR* and the newly designed *mreB* oligonucleotides was assessed. Thereafter, in view of the results obtained, the real-time PCR protocol was established and optimized using the *mreB* target gene. Finally, the detection and quantification of indigenous *V. harveyi* in seawater and biofilm from a fish farm were performed.

## 2. Materials and methods

### 2.1. Bacteria strains and culture methods

A total of 85 *Vibrio* strains were used in this study (Table 1): (i) 38 collection strains were obtained from the Belgian Coordinated Collections of Microorganisms (BCCM/LMG), the German Collection of Microorganisms and Cell Cultures (DSMZ), the Spanish Type Culture Collection (CECT), and the Collection of Institut Pasteur (CIP), (ii) 35 isolates were obtained from a fish farm raising seabass *Dicentrarchus labrax*. Bacteria were isolated from the spleen of moribund fish, plated on marine agar (MA), (Difco Laboratories, Detroit, MI, USA), incubated at 25 °C overnight and then identified by matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI TOF MS) using the Bruker Biotyper database by a veterinary laboratory, and (iii) 12 isolates were isolated by the French Research Institute for the Exploitation of the Sea (IFREMER) from oysters (*Crassostrea gigas*) and abalone (*Haliotis tuberculata*). All bacteria were maintained in Luria-Bertani broth supplemented with
20% NaCl (LBS), with a final pH of 7.2 ± 0.2, and with 20% glycerol and stored at -80 °C until use. Bacteria were plated on thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Biokar Diagnostics, Beauvais, France) and incubated overnight at growth temperature, in order to check the purity of the isolates. A single colony was then plated on LBS agar and incubated overnight at growth temperature. This temperature was 37 °C, except for LMG 11216T, LMG 25266T, Vh2, Vh3, Vt1, Vt2 and Vg1, for which the growth temperature was 25 °C.

<table><caption>Table 1. List of collection strains and bacterial isolates used in this study</caption><thead><tr><th rowspan="2">Strain/isolate ID</th><th rowspan="2">Assigned species</th><th rowspan="2">Host/Origin</th><th colspan="2">Isolation</th></tr><tr><th>Place</th><th>Date</th></tr></thead><tbody><tr><td>LMG 4044<sup>T</sup></td><td>Vibrio harveyi</td><td>Dead amphipod (Talorchestia sp.)</td><td>United States</td><td>/</td></tr><tr><td>LMG 7890</td><td>Vibrio harveyi</td><td>Brown shark (Carcharhinus plumbeus) kidney</td><td>United States</td><td>/</td></tr><tr><td>LMG 23680</td><td>Vibrio harveyi</td><td>Seawater</td><td>Japan</td><td>2005</td></tr><tr><td>LMG 19643</td><td>Vibrio harveyi</td><td>Japanese horse mackerel (Trachurus japonicus)</td><td>Japan</td><td>2002</td></tr><tr><td>LMG 23442</td><td>Vibrio harveyi</td><td>Dead abalone</td><td>Japan</td><td>2002</td></tr><tr><td>LMG 18299</td><td>Vibrio harveyi</td><td>Seabream (Sparus aurata)</td><td>Spain</td><td>1990</td></tr><tr><td>LMG 11755</td><td>Vibrio harveyi</td><td>Shark, mouth</td><td>Bahamas</td><td>/</td></tr><tr><td>LMG 22894</td><td>Vibrio harveyi</td><td>Shrimp, hemolymph</td><td>Mexico</td><td>1995</td></tr><tr><td>LMG 16832</td><td>Vibrio harveyi</td><td>Black tiger prawn</td><td>Thailand</td><td>/</td></tr><tr><td>LMG 22895</td><td>Vibrio harveyi</td><td>Shrimp, hepatopancreas</td><td>Mexico</td><td>1999</td></tr><tr><td>LMG 23678</td><td>Vibrio harveyi</td><td>Breeding seawater</td><td>Japan</td><td>2004</td></tr><tr><td>LMG 11226</td><td>Vibrio harveyi</td><td>/</td><td>/</td><td>/</td></tr><tr><td>LMG 23688</td><td>Vibrio harveyi</td><td>Abalone, internal organ</td><td>Japan</td><td>/</td></tr><tr><td>LMG 10946<sup>a</sup></td><td>Vibrio harveyi<sup>a</sup></td><td>Prawn (Palaemon indicus)</td><td>/</td><td>/</td></tr><tr><td>LMG 10947<sup>a</sup></td><td>Vibrio harveyi<sup>a</sup></td><td>Seawater</td><td>Red Sea</td><td>/</td></tr><tr><td>LMG 11216<sup>T</sup></td><td>Vibrio campbellii</td><td>Seawater (800 meters)</td><td>Hawaii</td><td>/</td></tr><tr><td>LMG 21363</td><td>Vibrio campbellii</td><td>Diseased Penaeus monodon juvenile</td><td>Philippines</td><td>/</td></tr><tr><td>LMG 11256</td><td>Vibrio campbellii</td><td>Seawater</td><td>Hawaii</td><td>/</td></tr><tr><td>CIP 70.67</td><td>Vibrio campbellii</td><td>/</td><td>/</td><td>/</td></tr><tr><td>LMG 16862</td><td>Vibrio campbellii</td><td>Oyster</td><td>Spain</td><td>1989</td></tr><tr><td>LMG 16863</td><td>Vibrio campbellii</td><td>Oyster</td><td>Spain</td><td>1989</td></tr><tr><td>DSMZ 23055<sup>T</sup></td><td>Vibrio owensii</td><td>Spiny lobster (Panulirus ornatus) Aquaculture</td><td>Australia</td><td>/</td></tr><tr><td>LMG 25430<sup>T</sup></td><td>Vibrio owensii</td><td>Mussismilia hispida</td><td>Brazil</td><td>/</td></tr><tr><td>LMG 20370</td><td>Vibrio owensii</td><td>White shrimp (Litopenaeus vannamei)</td><td>Ecuador</td><td>/</td></tr><tr><td>LMG 4043</td><td>Vibrio owensii</td><td>Seawater</td><td>Israel</td><td>/</td></tr><tr><td>LMG 11659</td><td>Vibrio owensii</td><td>Seawater</td><td>Hawaii</td><td>/</td></tr><tr><td>LMG 21456</td><td>Vibrio rotiferianus</td><td>Rotifer (Brachionus plicatilis) culture</td><td>Belgium</td><td>/</td></tr><tr><td>DSMZ 17186<sup>T</sup></td><td>Vibrio rotiferianus</td><td>Rotifer (Brachionus plicatilis) culture</td><td>Belgium</td><td>/</td></tr><tr><td>LMG 2850<sup>T</sup></td><td>Vibrio parahaemolyticus</td><td>Human, "Shirashu" food poisoning</td><td>Japan</td><td>/</td></tr><tr><td>LMG 16838</td><td>Vibrio parahaemolyticus</td><td>Penaeus orientalis</td><td>China</td><td>/</td></tr><tr><td>LMG 11650</td><td>Vibrio alginolyticus</td><td>Seawater</td><td>/</td><td>/</td></tr><tr><td>LMG 4409<sup>T</sup></td><td>Vibrio alginolyticus</td><td>Spoiled horse mackerel (Trachurus trachurus) causing food poisoning</td><td>Japan</td><td>/</td></tr><tr><td>LMG 10950</td><td>Vibrio natriegens</td><td>Oyster</td><td>United States</td><td>/</td></tr><tr><td>CECT 526<sup>T</sup></td><td>Vibrio natriegens</td><td>Salt marsh mud</td><td>United States</td><td>/</td></tr></tbody></table>
<table><thead><tr><th rowspan="2">Strain/isolate ID</th><th rowspan="2">Assigned species</th><th rowspan="2">Host/Origin</th><th colspan="2">Isolation</th></tr><tr><th>Place</th><th>Date</th></tr></thead><tbody><tr><td>LMG 25266<sup>T</sup></td><td>Vibrio azureus</td><td>Seawater</td><td>Japan</td><td></td></tr><tr><td>CECT 8524</td><td>Vibrio jasicida</td><td>Lutjanus guttatus</td><td>Mexico</td><td>2004</td></tr><tr><td>DSMZ 19137<sup>T</sup></td><td>Vibrio mytili</td><td>Mytilus edulis</td><td>Spain</td><td>2007</td></tr><tr><td>CECT 7298<sup>T</sup></td><td>Vibrio sinaloensis</td><td>Lutjanus guttatus</td><td>Mexico</td><td>2003</td></tr><tr><td>Vh1 to Vh33<br>(Vh27 and Vh30<br>do not exist)</td><td>Vibrio harveyi</td><td>Sea bass (Dicentrarchus labrax) spleen,<br>fish farm</td><td>France</td><td>2017</td></tr><tr><td>Vh34 and Vh35</td><td>Vibrio harveyi</td><td>Haliotis tuberculata, juvenile, hatchery<br>(Manche)</td><td>France</td><td>2004</td></tr><tr><td>Vh36</td><td>Vibrio harveyi</td><td>Haliotis tuberculata, adult, hatchery<br>(Manche)</td><td>France</td><td>2004</td></tr><tr><td>Vh37</td><td>Vibrio harveyi</td><td>Haliotis tuberculata, adult (Fermanville)</td><td>France</td><td>2005</td></tr><tr><td>Vh38</td><td>Vibrio harveyi</td><td>Haliotis tuberculata, adult (Omonville)</td><td>France</td><td>2005</td></tr><tr><td>Vh43</td><td>Vibrio harveyi</td><td>C. gigas, spat (Arcachon)</td><td>France</td><td>2007</td></tr><tr><td>Vh45</td><td>Vibrio harveyi</td><td>C. gigas, juvenile (Thau)</td><td>France</td><td>2007</td></tr><tr><td>Vh47 and Vh48</td><td>Vibrio harveyi</td><td>Haliotis tuberculata, juvenile (Aber<br>Wrac'h)</td><td>France</td><td>2014</td></tr><tr><td>Vh51</td><td>Vibrio harveyi</td><td>C. gigas</td><td>France</td><td>2007</td></tr><tr><td>Vh52 and Vh53</td><td>Vibrio harveyi</td><td>C. gigas, larva, hatchery (Vendée)</td><td>France</td><td>2008</td></tr><tr><td>Vt1 and Vt2</td><td>Vibrio tasmaniensis</td><td>Sea bass (Dicentrarchus labrax) spleen,<br>fish farm</td><td>France</td><td>2017</td></tr><tr><td>Vg1</td><td>Vibrio gigantis</td><td>Sea bass (Dicentrarchus labrax) spleen,<br>fish farm</td><td>France</td><td>2017</td></tr><tr><td>Vp1</td><td>Vibrio rotiferianus</td><td>Sea bass (Dicentrarchus labrax) spleen,<br>fish farm</td><td>France</td><td>2017</td></tr></tbody></table>

## 2.2. Isolation of bacteria from seawater samples

Seawater volumes of 1 L from aquaculture tanks were collected 20 cm below the water surface. Samples were maintained on ice during transport to the laboratory and processed within 2 h. They were filtered through 0.45 µm-pore-size nitrocellulose filters (Sartorius, Göttingen, Germany). If the samples contained many aggregates and filters clogged, several filters were used. They were then immersed in 25 mL of LBS with 20% glycerol and stored at -80 °C until analysis. Subsequently, samples were defrosted at ambient temperature and vortexed for 3 min in order to free bacteria from filters. Filters were removed and the liquid was centrifuged at 8000×g for 10 min. The cell pellet was resuspended in 25 mL of physiological water (0.9% NaCl) and

<footnote><sup>a</sup> LMG 10946 and LMG 10947 strains were initially identified as Vibrio harveyi species but our study revealed misidentification</footnote>

<footnote><sup>T</sup> Type strain</footnote>
centrifuged at 8000×g for 10 min. Supernatant was removed and DNA was extracted from cell pellets.

## 2.3. Isolation of bacteria from biofilm samples

Pieces of concrete (5 cm × 2.5 cm) were made with the same materials as the walls of aquaculture tanks and immersed 20 cm below the water surface in these tanks. The objective was to mimic the biofilm formation occurring on the walls of aquaculture tanks, on pieces of concrete that can easily be retrieved. The concrete pieces, still immersed in seawater, were maintained on ice during transport to the laboratory and processed within 2 h. Bacteria from biofilm were collected by swabbing 2.34 cm² of concrete surface using a sterile stainless-steel jig. The procedure used to remove biofilm from concrete has previously been described by Mougin et al. (2019), and was modified slightly. Briefly, two swabs (150C, Copan, Brescia, Italy) were used twice for each piece of concrete and immersed into 3 mL of LBS with 20% glycerol. The tube containing the two swabs was then stored at -80 °C until analysis. Subsequently, samples were defrosted at ambient temperature and vortexed for 3 min in order to free bacteria from swabs. Swabs were removed and the liquid was centrifuged at 8000×g for 10 min. The cell pellet was resuspended in 3 mL of physiological water (0.9% NaCl) and centrifuged at 8000×g for 10 min. Supernatant was removed and DNA was extracted from cell pellets.

## 2.4. DNA extraction

DNA templates were extracted from pure culture, isolated from LBS agar, using the DNeasy Blood&Tissue kit (Qiagen, Hilden, Germany) and an automated nucleic acid extractor QIAcube Connect (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The final elution volume was 100 µL. The extracted DNA was then stored at -20 °C until use.

In order to prepare DNA templates from environmental samples, five DNA extraction kits were tested: the DNeasy PowerWater kit (no. 1), the DNeasy PowerBiofilm kit (no. 2), the DNeasy Blood&Tissue kit (no. 3) (Qiagen, Hilden, Germany), the Extracta DNA Prep for PCR-Tissue kit (no. 4) (Quantabio, Beverly, MA, USA), and the Arcis DNA Sample Prep kit (no. 5) (Arcis
Biotechnology, Daresbury, United Kingdom). For each kit, the protocol was carried out according to the manufacturer's instructions. Extracted DNA was resuspended in a final volume of 100 µL and stored at -20 °C.

**2.5. Design of primers, specificity, and annealing temperature**

Three *V. harveyi*-specific pairs of primers were tested in this study (Table 2): two pairs of primers targeting the *topA* and *toxR* genes (Pascual *et al.*, 2010, Cano-Gomez *et al.*, 2015), and one newly designed pair of primers targeting the *mreB* gene (Table 2).

In order to design the *mreB* primers, two alignments were performed. The first alignment allowed the identification of conserved regions shared by *V. harveyi* strains: 69 *mreB* gene sequences of *V. harveyi*, obtained from the GenBank database (National Center for Biotechnology Information, NCBI), were aligned using Mega X software version 10.0.1 (supplemental data, appendix A). The second alignment allowed the identification of divergent regions between *V. harveyi* and *V. harveyi*-related species: 9 *mreB* gene sequences of *V. harveyi*-related species were aligned with one *mreB* gene sequence of *V. harveyi* using MultAlin (Corpet, 1988) (supplemental data, appendix B). The target sequence was chosen from the conserved region of *V. harveyi* and the divergent region between *V. harveyi* and *V. harveyi*-related species. *In silico* analyses for melting temperature (Tm) estimation, secondary structures, and potential dimer formation of *mreB*, *topA* and *toxR* were performed using Oligo Calculator version 3.27 (Kibbe, 2007) and Primer3Plus (Rozen et Skaletsky, 2000).

The annealing temperatures (Ta) of all sets of primers were optimized using a temperature gradient ranging from 55 °C to 65 °C with the iCycler™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) (data not shown).

The *in silico* specificity of the primers was verified using the nucleotide BLAST search program with the GenBank database (NCBI). The experimental specificity was evaluated using conventional PCR. Exclusivity and inclusivity tests were performed in triplicate with a panel of
V. harveyi (n=58) and non- V. harveyi organisms (n=27) (Table 1). Exclusivity was validated by a lack of PCR products, and inclusivity was validated by a single band and expected product sizes on gel electrophoresis.

All primers were synthetized by TIB MOLBIOL (TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany) and were suspended in nuclease-free water to reach a final concentration of 10 µM and stored at -20 °C.

<table><caption>Table 2. Nucleotide sequences and characteristics of primers used for conventional and real-time PCR amplification</caption><thead><tr><th>Gene</th><th>Target organism</th><th>Primer name</th><th>Primer sequence (5'-3')</th><th>Annealing temperature (Ta)</th><th>Product length (bp)</th><th>Reference</th></tr></thead><tbody><tr><td>mreB</td><td>V. harveyi</td><td>mreB11F<br>mreB9bisR</td><td>TGAAGCTGTGATCAACTACG<br>CCTGACAGTGGCTCTTGTAA</td><td>55 °C</td><td>215</td><td>/</td></tr><tr><td>topA</td><td>V. harveyi</td><td>Vh.topA-F<br>Vh.topA-R</td><td>TGGCGCAGCGTCTATACG<br>TATTTGTCACCGAACTCAGAACC</td><td>55 °C</td><td>121</td><td>(Cano-<br>Gomez et<br>al., 2015)</td></tr><tr><td>toxR</td><td>V. harveyi</td><td>toxRF1<br>toxRR1</td><td>GAAGCAGCACTCACCGAT<br>GGTGAAGACTCATCAGCA</td><td>55 °C</td><td>382</td><td>(Pang et<br>al., 2006)</td></tr><tr><td>ftsZ</td><td>Vibrio spp.</td><td>VftsZ75F<br>VftsZ800R</td><td>GCTGTTGAACACATGGTACG<br>GCACCAGCAAGATCGATATC</td><td>50 °C</td><td>750</td><td>(Sawabe et<br>al., 2007a)</td></tr><tr><td>recA</td><td>Vibrio spp.</td><td>VrecA130F<br>VrecA720R</td><td>GTCTACCAATGGGTCGTATC<br>GCCATTGTAGCTGTACCAAG</td><td>50 °C</td><td>600</td><td>(Sawabe et<br>al., 2007a)</td></tr><tr><td>gyrB</td><td>Vibrio spp.</td><td>gyrB 1<br>gyrB 2</td><td>AGCCAAACNAAAGAYAARYT<br>CGYARYTTRTCYGGRTTRTRYTC</td><td>55 °C</td><td>493</td><td>(Teh et al.,<br>2010)</td></tr><tr><td>16S<br>rRNA</td><td>Vibrio spp.</td><td>567F<br>680R</td><td>GGCGTAAAGCGCATGCAGGT<br>GAAATTCTACCCCCCTCTACAG</td><td>60 °C</td><td>113</td><td>(Thompson<br>et al.,<br>2004b)</td></tr><tr><td>16S<br>rRNA<br>V4V5</td><td>High<br>coverage of<br>bacteria<br>and<br>archaea</td><td>515FY<br>926R</td><td>GTGYCAGCMGCCGCGGTAA<br>CCGYCAATTYMTTTRAGTTT</td><td>50 °C</td><td>411</td><td>(Quince et<br>al., 2011,<br>Parada et<br>al., 2016)</td></tr></tbody></table>
## 2.6. Conventional PCR conditions

The PCR reaction mixture contained 2.5 µL of 10X PCR buffer, 0.5 µL of 10 mM dNTP, 0.5 µL of each primer (10 µM), 0.125 µL of 5 units.µL⁻¹ HotStarTaq DNA polymerase (Qiagen, Hilden, Germany), 2 µL of template DNA (equilibrated to 25 ng.µL⁻¹), and 18.875 µL of nuclease-free water to a final volume of 25 µL. The PCR reaction was run on a Thermal Cycler (Applied Biosystems, Forster City, CA, USA), under the following conditions: 5 min at 94 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at the optimized Ta, and 1 min at 72 °C. The final cycle was followed by an additional 7 min of extension at 72 °C. All PCR experiments were carried out in triplicate and contained a positive control (LMG 4044 *V. harveyi* DNA), a negative control (CIP 70.67 *V. campbellii* DNA), and a no template control (NTC), DNA-free. The size of the PCR product was verified by ethidium bromide agarose gel electrophoresis (2%).

## 2.7. Real-time PCR conditions

The reaction mixture contained 10 µL of 2X Master Mix LightCycler® 480 SYBR Green I Master (Roche Diagnostics, France), 1 µL of each *mreB* primer (10 µM), 5 µL of template DNA, and 3 µL of water to a final volume of 20 µL. The quantitative PCR reaction was run on a LightCycler® 480 (Roche Diagnostics, France), under the following conditions: 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 20 s at 55 °C, and 10 s at 72 °C. The melting curve analysis was then performed under the following conditions: 5 s at 95 °C, 1 min at 65 °C, and an increase of the temperature from 65 °C to 97 °C at 0.3 °C.s⁻¹. All quantitative PCR experiments were performed in triplicate and included a positive control (LMG 4044 *V. harveyi* DNA), a negative control (CIP 70.67 *V. campbellii* DNA), and a no template control (NTC), DNA-free. Lack of specific amplification was defined for C~T~ values ≥ 35. The baseline of PCR was automatically set by the system, and data analysis was carried out with LightCycler® 480 SW 1.5.1 software.

## 2.8. Sequencing conditions

PCR amplification products were generated for three pairs of primers targeting the *ftsZ* (cell division protein FtsZ gene), *recA* (recombination and DNA repair protein gene) and *gyrB*
(uridylate kinase gene) genes (Table 2), screened from the literature (Sawabe et al., 2007a, Teh et al., 2010). The PCR reaction mixture contained 25 µL of 2X Platinum™ Green Hot Start PCR Master Mix (Invitrogen, Carlsbad, CA, USA), 1 µL of each primer (10 µM), 5 µL of template DNA (equilibrated to 25 ng.µL⁻¹), and 18 µL of nuclease-free water to a final volume of 50 µL. The PCR reaction was run on a Thermal Cycler (Applied Biosystems, Forster City, CA, USA), under the following conditions: 5 min at 95 °C, followed by 30 cycles of 1 min at 95 °C, 2 min 15 s at the optimized Ta, and 1 min 15 s at 72 °C. The final cycle was followed by an additional 7 min of extension at 72 °C. The PCR products of expected size were then sequenced from both sides (forward and reverse), using Sanger sequencing by GenoScreen (Lille, France). Analysis was performed using the nucleotide BLAST search program with the GenBank database (NCBI). For whole-genome sequencing, organisms were sent to GenoScreen on LBS agar after incubation at growth temperature. Analysis was performed with CLC Genomics Workbench version 20.0.2. Raw sequences were paired and trimmed using default parameters and then assembled into contigs using the default parameters of the Genome Finishing Module. A distance tree was generated using the neighbor joining algorithm, with these contigs and different genome sequences of *Vibrio* spp. available on NCBI.

## 2.9. Real-time PCR validation

For the sensitivity of the DNA detection assay, pure *V. harveyi* DNA was extracted and serially diluted in nuclease-free water. The purity and the quantity of the extracted DNA were determined using a DeNovix DS-11 spectrophotometer (Clinisciences, Nanterre, France) and a Qubit 3.0 fluorometer (dsDNA high-sensitivity assay), (Invitrogen, Carlsbad, CA, USA), respectively. The number of genome copies was estimated from the DNA quantity measured, considering the genome size of *V. harveyi* equal to 5.67×10⁶ bp (Brankatschk et al., 2012, Urbanczyk et al., 2013b). The calibration curve was plotted using the means of C~T~ values of 20 replicate series versus the logarithm of each dilution factor.

The presence of PCR inhibitors in the extracted DNA templates was verified by adding an amount of 125 ng of pure *V. harveyi* DNA into template DNA obtained from 3 different seawater
and 3 different biofilm samples, in order to reach a final concentration of 10⁶ genome copies per reaction. A 10-fold dilution series of inoculated DNA template and pure DNA, prepared in nuclease-free water, were then amplified by real-time PCR.

In order to verify whether the assay enabled detection and quantification of indigenous *V. harveyi* from environmental samples, real-time analysis of 4 seawater and 4 biofilm samples collected from a fish farm was performed. Genome copy equivalent (GE) concentrations were estimated by standard curve, taking into account the dilution factor of the assay.

# 3. Results

## 3.1. Specificity of the real-time PCR assay

First, the exclusivity of the *mreB*, *topA* and *toxR* primers was confirmed by performing conventional PCR on a panel of 23 non-*V. harveyi* collection strains including, at least one reference strain for each of the 10 species, and 4 non-*V. harveyi* (Table 1). These organisms were mostly species in the Harveyi clade, genetically close to *V. harveyi*. Second, the inclusivity of the three pairs of primers was determined by testing a panel of 15 *V. harveyi* collection strains, including the reference strain LMG 4044T. Then, 43 *V. harveyi* isolates obtained from reared seabass (n=31), abalone (*H. tuberculata*) (n=7), and oyster (*C. gigas*) (n=5) were tested. Among these 58 organisms, only 53, 52 and 48 showed positive results for the *mreB*, *topA* and *toxR* primers, respectively (Table 3.A). In fact, for all the primers, no PCR product was detected from the LMG 10946 and LMG 10947 collection strains and from the Vh2, Vh15 and Vh28 isolates. Moreover, the results diverged between the *mreB*, *topA* and *toxR* primers for 5 isolates. PCR products were detected from Vh5, Vh8, Vh22 and Vh29 isolates using the *mreB* and *topA* primers, while no PCR product was detected using the *toxR* primers. PCR products were detected from the Vh25 isolate using the *mreB* primers, while no PCR product was detected using the *topA* and *toxR* primers. As misidentifications could have occurred, identification of these 10 organisms required confirmation.
In order to clearly identify these 10 organisms (LMG 10946, LMG 10947, Vh2, Vh5, Vh8, Vh15, Vh22, Vh25, Vh28 and Vh29), Sanger sequencing was performed targeting three reference genes: *ftsZ*, *recA*, and *gyrB*. The non-*V. harveyi* isolates Vt1, Vt2, Vg1 and Vp1 were also sequenced. When the sequencing results were divergent between the three genes, whole-genome sequencing was performed. Analysis of Vh5, Vh8, Vh22, Vh25 and Vh29 sequences showed high identity to *V. harveyi* (supplemental data, appendix C). However, LMG 10946, LMG 10947, Vh2, Vh15, Vh28, Vt1, Vt2, Vg1 and Vp1 sequences exhibited high identity to non-*V. harveyi* species (supplemental data, appendix C and D). PCR results obtained with the *mreB* primers fitted with these data, whereas those obtained with *topA* and *toxR* did not (Table 3.B).

Ultimately, the *mreB*, *topA* and *toxR* primers exhibited 100%, 98.1% and 90.6% inclusivity, respectively. The lack of PCR product detected from all the non-*V. harveyi* strains (n = 27) revealed 100% exclusivity for all primers.

The specificity of the *mreB* primers was confirmed by sequencing PCR products obtained from the LMG 4044T reference strain. Moreover, analysis of the melting curve, obtained from real-time PCR experiments, highlighted only one peak with a melting temperature (Tm) of 83.70 ± 0.10 °C (data not shown). Detection of PCR products using 16S rRNA primers from all tested strains confirmed that DNA extraction was properly performed. The inclusion of blank samples with no template DNA confirmed non-specific reactions between the components.
<table><caption>Table 3. Specificity of the *mreB*, *topA* and *toxR* primers according to collection strains and bacterial isolates used in the present study. A. PCR results of the 85 organisms studied. B. Detail of the PCR and sequencing results of the 10 *V. harveyi* strains that showed divergence with initially assigned species identification</caption><caption>A.</caption><thead><tr><th rowspan="2">Species</th><th rowspan="2">Number of strains</th><th colspan="4">PCR results</th></tr><tr><th>215 bp<br><i>mreB</i></th><th>121 bp<br><i>topA</i></th><th>382 bp<br><i>toxR</i></th><th>113 bp<br>16S rRNA</th></tr></thead><tbody><tr><td><i>V. harveyi</i></td><td>58</td><td>+(53)</td><td>+(52)</td><td>+(48)</td><td>+(58)</td></tr><tr><td><i>V. campbellii</i></td><td>6</td><td>-</td><td>-</td><td>-</td><td>+(6)</td></tr><tr><td><i>V. owensii</i></td><td>5</td><td>-</td><td>-</td><td>-</td><td>+(5)</td></tr><tr><td><i>V. rotiferianus</i></td><td>3</td><td>-</td><td>-</td><td>-</td><td>+(3)</td></tr><tr><td><i>V. parahaemolyticus</i></td><td>2</td><td>-</td><td>-</td><td>-</td><td>+(2)</td></tr><tr><td><i>V. alginolyticus</i></td><td>2</td><td>-</td><td>-</td><td>-</td><td>+(2)</td></tr><tr><td><i>V. natriegens</i></td><td>2</td><td>-</td><td>-</td><td>-</td><td>+(2)</td></tr><tr><td><i>V. azureus</i></td><td>1</td><td>-</td><td>-</td><td>-</td><td>+(1)</td></tr><tr><td><i>V. jasicida</i></td><td>1</td><td>-</td><td>-</td><td>-</td><td>+(1)</td></tr><tr><td><i>V. mytili</i></td><td>1</td><td>-</td><td>-</td><td>-</td><td>+(1)</td></tr><tr><td><i>V. sinaloensis</i></td><td>1</td><td>-</td><td>-</td><td>-</td><td>+(1)</td></tr><tr><td><i>V. tasmaniensis</i></td><td>2</td><td>-</td><td>-</td><td>-</td><td>+(2)</td></tr><tr><td><i>V. gigantis</i></td><td>1</td><td>-</td><td>-</td><td>-</td><td>+(1)</td></tr></tbody></table>

<table><caption>B.</caption><thead><tr><th>Assigned species</th><th>Strain/isolate ID</th><th>Sequencing identification results</th><th></th><th></th></tr></thead><tbody><tr><td><i>V. harveyi</i></td><td>LMG 10946</td><td><i>V. campbellii</i></td><td>-</td><td>-</td></tr><tr><td><i>V. harveyi</i></td><td>LMG 10947</td><td><i>V. owensii</i></td><td>-</td><td>-</td></tr><tr><td><i>V. harveyi</i></td><td>Vh2</td><td><i>V. jasicida</i></td><td>-</td><td>-</td></tr><tr><td><i>V. harveyi</i></td><td>Vh5</td><td><i>V. harveyi</i></td><td>+</td><td>+</td></tr><tr><td><i>V. harveyi</i></td><td>Vh8</td><td><i>V. harveyi</i></td><td>+</td><td>+</td></tr><tr><td><i>V. harveyi</i></td><td>Vh15</td><td><i>V. rotiferianus</i></td><td>-</td><td>-</td></tr><tr><td><i>V. harveyi</i></td><td>Vh22</td><td><i>V. harveyi</i></td><td>+</td><td>+</td></tr><tr><td><i>V. harveyi</i></td><td>Vh25</td><td><i>V. harveyi</i></td><td>+</td><td>-</td></tr><tr><td><i>V. harveyi</i></td><td>Vh28</td><td><i>V. owensii</i></td><td>-</td><td>-</td></tr><tr><td><i>V. harveyi</i></td><td>Vh29</td><td><i>V. harveyi</i></td><td>+</td><td>+</td></tr></tbody></table>

+' Positive, '- Negative

## 3.2. Sensitivity and quantification limits of the assay

Assay sensitivity was determined using 10 independent dilution series of purified LMG 4044T *V. harveyi* DNA, ranging from 2, 5, and 10 to 10⁷ genome copies per reaction, in duplicate. The lowest number of genome copies detected in at least 95% of the 20 replicates was 5 genome copies per reaction, corresponding to the limit of detection (LOD) for pure DNA of *V. harveyi*.
The linear regression showed accurate correlation (R=0.9907) between the logarithm of genome
copies per reaction and the threshold cycle (C<sub>T</sub>) value (Fig. 1). The correlation was linear over a
range of 5 to 10<sup>7</sup> genome copies per reaction, confirming that the limit of quantification (LOQ)
corresponded to 5 genome copies per reaction. Real-time PCR efficiency was calculated from the
slope of the linear portion of the calibration curve, according to the formula: E = 10<sup>-1/slope</sup> - 1 and
was equal to 99.9%.

<figure><img src="image_34.png" /><figcaption>Figure 1. Standard curve representing the detection and quantification of *V. harveyi* DNA by real-time PCR targeting the *mreB* gene. Means of 20 replicates are represented with corresponding standard deviation</figcaption></figure>

**3.3. Quantitative detection of *V. harveyi* in environmental samples**

In order to obtain DNA templates with optimal quantity and purity from both seawater and
biofilm samples, five extraction kits were compared (Table 4). To check the integrity of extracted
DNA and the potential presence of PCR inhibitors, two conventional PCRs were carried out.
The first targeted the 16S rRNA V4V5 hypervariable region found in most bacteria and archaea,
and the second targeted 16S rRNA found in most *Vibrio* species. Both PCR analyses indicated
the potential presence of PCR inhibitors when using no. 3 and no. 1 kits. In fact, no PCR product
was detected from seawater and biofilm DNA templates extracted with the no. 3 kit. PCR
products were only detected from seawater DNA templates extracted with the no. 1 kit using
16S rRNA V4V5 primers. These results highlighted that the no. 3 kit was not suitable for DNA
extraction from either of our samples, and the no. 1 kit was not suitable for DNA extraction from biofilm samples. The seawater and biofilm DNA templates extracted with no. 4 and no. 5 kits were cloudy solutions. That is why the PCR analyses were not carried out and the two kits were not selected. PCR products were detected from seawater and biofilm extracted DNA templates with the no. 2 kit, using both primers. Moreover, the highest quantity of extracted DNA was obtained with the no. 2 kit. These results indicated that the no. 2 kit – DNeasy PowerBiofilm kit (Qiagen) – is suitable for the extraction of Vibrio DNA from both our environmental samples and was therefore selected.

<table><caption>Table 4. Comparison of the DNA extraction methods for both matrices seawater and biofilm samples. Values are the means of two independent replicates.</caption><thead><tr><th rowspan="2">No.</th><th rowspan="2">DNA extraction kit</th><th rowspan="2">Matrix</th><th>Qubit</th><th colspan="2">DeNovix</th><th colspan="2">PCR</th></tr><tr><th>DNA ng.µL<sup>-1</sup></th><th>DNA ng.µL<sup>-1</sup></th><th>OD 260/230</th><th>OD 260/280</th><th>16S rRNA V4V5 All bacteria</th><th>16S rRNA Vibrio spp.</th></tr></thead><tbody><tr><td rowspan="2">1</td><td rowspan="2">DNeasy PowerWater kit Qiagen</td><td>Seawater</td><td>3.73</td><td>10.65</td><td>0.54</td><td>1.10</td><td>+</td><td>-</td></tr><tr><td>Biofilm</td><td>2.82</td><td>6.87</td><td>0.34</td><td>0.67</td><td>-</td><td>-</td></tr><tr><td rowspan="2">2</td><td rowspan="2">DNeasy PowerBiofilm kit Qiagen</td><td>Seawater</td><td>10.56</td><td>17.75</td><td>0.80</td><td>1.45</td><td>+</td><td>+</td></tr><tr><td>Biofilm</td><td>6.15</td><td>8.90</td><td>0.24</td><td>1.07</td><td>+</td><td>+</td></tr><tr><td rowspan="2">3</td><td rowspan="2">DNeasy Blood&amp;Tissue kit Qiagen</td><td>Seawater</td><td>2.07</td><td>11.04</td><td>0.51</td><td>2.18</td><td>-</td><td>-</td></tr><tr><td>Biofilm</td><td>2.73</td><td>8.25</td><td>0.87</td><td>1.49</td><td>-</td><td>-</td></tr><tr><td rowspan="2">4</td><td rowspan="2">Extracta DNA Prep for PCR-Tissue kit Quantabio</td><td>Seawater</td><td>Too low</td><td>231.75</td><td>0.59</td><td>1.35</td><td></td><td></td></tr><tr><td>Biofilm</td><td>Too low</td><td>242.04</td><td>0.59</td><td>1.63</td><td></td><td></td></tr><tr><td rowspan="2">5</td><td rowspan="2">Arcis DNA Sample Prep kit Arcis Biotechnology</td><td>Seawater</td><td>3.74</td><td>208.07</td><td>0.14</td><td>0.68</td><td></td><td></td></tr><tr><td>Biofilm</td><td>3.85</td><td>216.88</td><td>0.14</td><td>0.69</td><td></td><td></td></tr></tbody></table>

OD, optic density

Even though this chosen kit seemed to reduce the amount of co-extracted inhibitors, some of them could still subsist in the environmental extracted DNA templates. This is why the effect of these inhibitors was evaluated. A known amount of pure V. harveyi DNA was added to 3 different
seawater and 3 different biofilm DNA templates. These DNA templates were extracted with the no. 2 kit, and previously identified as negative for the target gene. A series of 10-fold dilutions, ranging from 10² to 10⁶ genome copies per reaction of pure DNA template, was prepared and used as a standard. Series of 10-fold dilutions (10² to 10⁶ genome copies per reaction) of inoculated seawater and biofilm DNA templates were then prepared and amplified by real-time PCR. The results showed a significant increase in the C~T~ value for the less diluted seawater and biofilm DNA templates (10⁶) compared to the C~T~ value of pure DNA (Fig. 2). The data obtained confirmed that seawater and biofilm samples contained inhibitors affecting assay efficiency. Nevertheless, the results indicated that the effect of inhibitors was avoided by 10-fold dilution since no increase in C~T~ value was observed for the 10-fold diluted DNA template (10² to 10⁵). Therefore, the DNA templates extracted from both samples must be 10-fold diluted in order to avoid the influence of real-time PCR inhibitors.

<figure><img src="image_35.png" /><figcaption>Figure 2. Plot of 10-fold dilution series representing the detection and quantification of *V. harveyi* DNA by real-time PCR from pure culture of *V. harveyi* and from inoculated seawater and biofilm DNA templates</figcaption></figure>

The real-time PCR assay was validated by detecting and quantifying indigenous *V. harveyi* by real-time PCR from 4 seawater and 4 biofilm samples, collected from a seabass farm. The results showed specific amplification from both seawater and biofilm samples (Table 5). However, a negative result was obtained from one biofilm sample, meaning that the quantity of *V. harveyi*
was below the limit of detection of the assay. The concentration of indigenous *V. harveyi* ranged from 1.23 to 2.68×10² GE.mL⁻¹ in seawater, and from 2.84×10² to 5.00×10³ GE.cm⁻² in biofilm samples.

<table><caption>Table 5. Quantification of *V. harveyi* from seawater and biofilm samples collected from a seabass farm</caption><thead><tr><th>Date</th><th>Sample</th><th>Estimated quantity in samples</th></tr></thead><tbody><tr><td>May 3, 2018</td><td>Seawater</td><td>5.82 × 10<sup>1</sup> GE.mL<sup>-1</sup></td></tr><tr><td></td><td>Biofilm</td><td>ND</td></tr><tr><td>Jul. 11, 2018</td><td>Seawater</td><td>2.10 × 10<sup>2</sup> GE.mL<sup>-1</sup></td></tr><tr><td></td><td>Biofilm</td><td>1.04 × 10<sup>3</sup> GE.cm<sup>-2</sup></td></tr><tr><td>Aug. 22, 2018</td><td>Seawater</td><td>2.68 × 10<sup>2</sup> GE.mL<sup>-1</sup></td></tr><tr><td></td><td>Biofilm</td><td>5.00 × 10<sup>3</sup> GE.cm<sup>-2</sup></td></tr><tr><td>Oct. 17, 2018</td><td>Seawater</td><td>1.23 × 10<sup>0</sup> GE.mL<sup>-1</sup></td></tr><tr><td></td><td>Biofilm</td><td>2.84 × 10<sup>2</sup> GE.cm<sup>-2</sup></td></tr></tbody></table>

ND, not detected

GE, genome equivalent

## 4. Discussion

As *Vibrio harveyi* is a major pathogen in aquaculture, its early detection and quantification are a key focus for aquaculture farms. Development of reliable identification tools is complex since species belonging to the Harveyi clade are genetically and phenotypically similar. Whole-genome and fingerprinting techniques exhibited high discriminatory power. Nevertheless, these later are expensive, time-consuming and thus not suitable to provide a rapid diagnosis in a context of vibriosis outbreaks (Cano-Gomez et al., 2009). On the contrary, real-time PCR is a rapid and reliable method that is commonly used. Previous studies have highlighted the need for more *V. harveyi*-specific primers and accurate detection sensitivity in order to prevent vibriosis in aquaculture facilities (Fukui et Sawabe, 2008, Schikorski et al., 2013).

In this work, a *V. harveyi*-specific quantitative real-time PCR was developed. The fluorescent reporter SYBR Green I was used since it has the advantage of not involving the design of an oligonucleotide probe. The protocol was established with a newly designed pair of primers targeting the *mreB* gene, which is a single-copy gene that is stable in the genome (Cano-Gomez et al., 2011). The present real-time PCR allowed for discrimination between *V. harveyi* and
closely related species belonging to the Harveyi clade, such as *V. alginolyticus*, *V. azureus*, *V. campbellii*, *V. jasicida*, *V. mytili*, *V. natriegens*, *V. owensii* (synonym of *V. communis*), *V. parahaemolyticus*, and *V. rotiferianus* (Sawabe et al., 2007a, Hoffmann et al., 2012, Urbanczyk et al., 2013b).

Additional information was found regarding the exclusivity and inclusivity of the *topA* and *toxR* primers previously designed by Cano-Gomez et al. (2015) and Pang et al. (2006), respectively. The lack of inclusivity of both sets of primers suggested sequence heterogeneities in the *topA* and *toxR* genes of epidemiologically distinct strains within *V. harveyi* species. Importantly, *Vibrio* species are known for high genomic plasticity (Rowe-Magnus et al., 2006). This is consistent with the study carried out by Conejero et Hedreyda (2003), who developed a *V. harveyi*-specific conventional PCR targeting the *toxR* gene. The validation experiments revealed false-negative results for two *V. harveyi* isolates: STD 3-101 and VIB 391. Both these organisms were isolated from shrimps while other organisms that showed positive results were isolated from fishes. This shows that the high genomic plasticity of *V. harveyi* can lead to false-negative results. Nevertheless, in the present study, the *mreB* gene appeared relatively well conserved since no false-negative results were recorded for our isolates.

Discriminating between species belonging to the Harveyi clade is complex and misidentifications can occur. Our results showed that the *V. harveyi*-LMG 10946 and LMG 10947 collection strains have previously been identified incorrectly. According to our data, LMG 10946 was identified as *V. campbellii* and LMG 10947 as *V. owensii*. Thompson et al. (2001) had studied both strains, among others, using fluorescent amplified fragment length polymorphism (FAFLP) genotyping. The strains were distributed in the same cluster as the LMG 4043 and LMG 11659 collection strains identified as *V. owensii*. Therefore, these results indicated genome similarities with non-*V. harveyi* species, suggesting potential misidentification. Furthermore, Hoffmann et al. (2012) reported previous incorrect identifications for several collection strains, by performing an MLSA study based on the *ftsZ*, *mreB*, *rctB*, *rpoD*, *topA* and *toxR* genes. They confirmed that strains LMG 16862 and LMG 16863, first identified as *V. harveyi*, were actually *V. campbellii*, and LMG
4043 was *V. owensii*. They also demonstrated that *V. communis* and *V. owensii* are synonyms.
In our study, negative PCR results were obtained using the *mreB*, *topA* and *toxR* primers for all
the collection strains previously mentioned, confirming the accurate exclusivity of all sets of
primers.

The present study clearly demonstrates the complexities of processing environmental samples
from aquaculture systems. Our method was optimized to quantify the population of indigenous
*V. harveyi* in complex environmental samples: seawater and biofilm from aquaculture. Detection
of target DNA in tanks was even more problematic due to the high diversity and abundance of
organic matter. The challenge was to provide accurate DNA yield, while preserving DNA quality.
Filtration is a common method used to process aquatic samples (Staley et al., 2015). This method
makes it possible to concentrate bacteria and thereby increase DNA yield, without previous
enrichment steps, which can be time-consuming (Akkermans et al., 1995). Likewise, swabbing
has been widely used to collect environmental biofilm samples from concrete (De Muynck et al.,
2010). However, environmental samples often contain various PCR-inhibitors leading to false-
negative results or inaccurate quantification (Wilson, 1997). That is why our study reports the
DNA extraction efficiency for five distinct DNA extractions kits. We found that the DNA
extraction kit allowing the best DNA yield and purity was the DNeasy PowerBiofilm kit
(Qiagen), which was therefore selected to perform the assay. Nevertheless, this optimization was
not sufficient and a few inhibitors remained in extracted template DNA. The effects of these
inhibitors can be avoided by a simple widely used method, which consists in diluting template
DNA of environmental samples (Wilson, 1997). However, the target DNA is also diluted, and
this strategy reduces detection sensitivity. The real-time PCR detection limit for *V. harveyi*
derived from pure culture was 5 genome copies per PCR reaction. Although complex
environmental matrices can affect sensitivity, the assay detection limit for environmental samples
could not be determined due to background *V. harveyi* levels in seawater and biofilm samples.
However, performing this assay in aquaculture systems did enable the detection and
quantification of indigenous *V. harveyi* in unseeded samples. The present method is therefore
applicable in aquaculture facilities.
The concentration of indigenous *V. harveyi* detected in seawater samples ranged from 1 to 10² GE.mL⁻¹. Higher bacteria abundances were obtained in the study carried out by Zhou *et al.* (2007). They quantified the abundance of *V. alginolyticus* in environmental seawater by real-time PCR. The concentration ranged from 10² to 10³ CFU.mL⁻¹. Likewise, Saulnier *et al.* (2009) quantified the abundance of *V. aestuarianus* in seawater by real-time PCR. The concentrations found ranged from 10¹ to 10² cells.mL⁻¹. The volume of the treated sample could explain this difference. Importantly, in these previous studies, volumes of 1 mL or less of seawater were analyzed by real-time PCR, while in the present study, 1 L of seawater was first concentrated and then analyzed. In this way, the concentration of bacteria by filtration allowed us to improve the detection limit. Nevertheless, the concentration of large volumes of water can lead to a significant loss of the target organism during the filtration procedure (Akkermans *et al.*, 1995, Fukui et Sawabe, 2008). The concentration of indigenous *V. harveyi* detected in biofilm samples ranged from 10¹ to 10³ GE.cm⁻². This value makes sense when compared to the findings reported by Shikuma et Hadfield (2010). They quantified the abundance of *V. cholerae* in seawater and biofilm samples isolated from harbors by real-time PCR. High abundance of *V. cholerae* was detected and the concentration obtained was around 10³ GE.cm⁻². They also highlighted that the concentration in seawater samples was lower than in biofilm, and was approximately 10¹ GE.mL⁻¹. Thus, our method allows for accurate quantification of bacteria in environmental samples.

**5. Conclusion**

To conclude, the present method is a useful molecular tool, allowing for direct quantification of *V. harveyi* in seawater and biofilm samples from aquaculture. The *mreB* primers designed showed high specificity for *V. harveyi* strains isolated from various organisms, achieving discrimination between *V. harveyi* and closely related species belonging to the Harveyi clade. This tool will be used in a forthcoming study with the aim of monitoring seasonal changes in *V. harveyi* abundance in aquaculture facilities.
## Funding

Julia Mougin would like to thank the Hauts-de-France regional council and ULCO for their financial support of her PhD studies. This work was funded by the French government, Ifremer and the region Hauts-de-France in the framework of the CPER 2014-2020 MARCO project. This work was also supported by a grant from FEAMP (PFEA470017FA1000006) as part of the LUVIBAR project.

## Acknowledgements

The authors thank Aquanord-Ichtus as a contributor to the LUVIBAR project and especially Guillaume Tielie, Anabelle Duhamel and Céline Doyen. The authors are also grateful to Thomas Brauge and Stéphanie Copin for their scientific assistance. The authors thank the National Reference Laboratory for mollusk diseases for sharing *V. harveyi* strains, and the French ministry DGAL founding the NRL. Finally, the authors thank the veterinary laboratory LABOCEA for sharing *V. harveyi* strains isolated from Aquanord-Ichtus.

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Supplemental data

Appendix A. List of *mreB* sequences of *V. harveyi*, aligned in order to identify conserved regions shared by *V. harveyi* strains.

<table><thead><tr><th>No.</th><th>Species</th><th>GenBank accession no.</th></tr></thead><tbody><tr><td>1</td><td>Vibrio harveyi</td><td>JNO33881.1</td></tr><tr><td>2</td><td>Vibrio harveyi</td><td>LC370163.1</td></tr><tr><td>3</td><td>Vibrio harveyi</td><td>LC370156.1</td></tr><tr><td>4</td><td>Vibrio harveyi</td><td>LC071934.1</td></tr><tr><td>5</td><td>Vibrio harveyi</td><td>LC370168.1</td></tr><tr><td>6</td><td>Vibrio harveyi</td><td>LC370167.1</td></tr><tr><td>7</td><td>Vibrio harveyi</td><td>LC370166.1</td></tr><tr><td>8</td><td>Vibrio harveyi</td><td>LC370165.1</td></tr><tr><td>9</td><td>Vibrio harveyi</td><td>LC370162.1</td></tr><tr><td>10</td><td>Vibrio harveyi</td><td>LC370161.1</td></tr><tr><td>11</td><td>Vibrio harveyi</td><td>LC370159.1</td></tr><tr><td>12</td><td>Vibrio harveyi</td><td>LC370158.1</td></tr><tr><td>13</td><td>Vibrio harveyi</td><td>LC370157.1</td></tr><tr><td>14</td><td>Vibrio harveyi</td><td>LC370151.1</td></tr><tr><td>15</td><td>Vibrio harveyi</td><td>LC370129.1</td></tr><tr><td>16</td><td>Vibrio harveyi</td><td>LC370164.1</td></tr><tr><td>17</td><td>Vibrio harveyi</td><td>LC370143.1</td></tr><tr><td>18</td><td>Vibrio harveyi</td><td>LC071932.1</td></tr><tr><td>19</td><td>Vibrio harveyi</td><td>LC370152.1</td></tr><tr><td>20</td><td>Vibrio harveyi</td><td>LC370150.1</td></tr><tr><td>21</td><td>Vibrio harveyi</td><td>LC370145.1</td></tr><tr><td>22</td><td>Vibrio harveyi</td><td>LC370144.1</td></tr><tr><td>23</td><td>Vibrio harveyi</td><td>LC370141.1</td></tr><tr><td>24</td><td>Vibrio harveyi</td><td>LC370139.1</td></tr><tr><td>25</td><td>Vibrio harveyi</td><td>LC370136.1</td></tr><tr><td>26</td><td>Vibrio harveyi</td><td>LC370135.1</td></tr><tr><td>27</td><td>Vibrio harveyi</td><td>LC370128.1</td></tr><tr><td>28</td><td>Vibrio harveyi</td><td>LC370160.1</td></tr><tr><td>29</td><td>Vibrio harveyi</td><td>LC370148.1</td></tr><tr><td>30</td><td>Vibrio harveyi</td><td>LC370142.1</td></tr><tr><td>31</td><td>Vibrio harveyi</td><td>CP025537.1</td></tr><tr><td>32</td><td>Vibrio harveyi</td><td>DQ907452.1</td></tr><tr><td>33</td><td>Vibrio harveyi</td><td>JF930444.1</td></tr><tr><td>34</td><td>Vibrio harveyi</td><td>GQ428275.1</td></tr><tr><td>35</td><td>Vibrio harveyi</td><td>DQ907423.1</td></tr><tr><td>36</td><td>Vibrio harveyi</td><td>JF930449.1</td></tr><tr><td>37</td><td>Vibrio harveyi</td><td>JF930448.1</td></tr><tr><td>38</td><td>Vibrio harveyi</td><td>JF930445.1</td></tr><tr><td>39</td><td>Vibrio harveyi</td><td>JF930443.1</td></tr><tr><td>40</td><td>Vibrio harveyi</td><td>DQ907422.1</td></tr><tr><td>41</td><td>Vibrio harveyi</td><td>JF930447.1</td></tr><tr><td>42</td><td>Vibrio harveyi</td><td>GQ428272.1</td></tr><tr><td>43</td><td>Vibrio harveyi</td><td>GQ428271.1</td></tr><tr><td>44</td><td>Vibrio harveyi</td><td>GQ428270.1</td></tr><tr><td>45</td><td>Vibrio harveyi</td><td>GQ428253.1</td></tr><tr><td>46</td><td>Vibrio harveyi</td><td>GQ428258.1</td></tr><tr><td>47</td><td>Vibrio harveyi</td><td>GQ428262.1</td></tr></tbody></table>
<table><thead><tr><td>No.</td><td>Species</td><td>GenBank accession no.</td></tr></thead><tbody><tr><td>48</td><td>Vibrio harveyi</td><td>GQ428260.1</td></tr><tr><td>49</td><td>Vibrio harveyi</td><td>GQ428251.1</td></tr><tr><td>50</td><td>Vibrio harveyi</td><td>GQ428265.1</td></tr><tr><td>51</td><td>Vibrio harveyi</td><td>GQ428259.1</td></tr><tr><td>52</td><td>Vibrio harveyi</td><td>GQ428256.1</td></tr><tr><td>53</td><td>Vibrio harveyi</td><td>GQ428261.1</td></tr><tr><td>54</td><td>Vibrio harveyi</td><td>HQ449927.1</td></tr><tr><td>55</td><td>Vibrio harveyi</td><td>AB428991.1</td></tr><tr><td>56</td><td>Vibrio harveyi</td><td>GQ428264.1</td></tr><tr><td>57</td><td>Vibrio harveyi</td><td>HQ449929.1</td></tr><tr><td>58</td><td>Vibrio harveyi</td><td>HQ449926.1</td></tr><tr><td>59</td><td>Vibrio harveyi</td><td>HQ449925.1</td></tr><tr><td>60</td><td>Vibrio harveyi</td><td>HQ449930.1</td></tr><tr><td>61</td><td>Vibrio harveyi</td><td>HQ449929.1</td></tr><tr><td>62</td><td>Vibrio harveyi</td><td>GQ428254.1</td></tr><tr><td>63</td><td>Vibrio harveyi</td><td>GQ428250.1</td></tr><tr><td>64</td><td>Vibrio harveyi</td><td>GQ428252.1</td></tr><tr><td>65</td><td>Vibrio harveyi</td><td>HQ540725.1</td></tr><tr><td>66</td><td>Vibrio harveyi</td><td>GQ428255.1</td></tr><tr><td>67</td><td>Vibrio harveyi</td><td>GQ496611.1</td></tr><tr><td>68</td><td>Vibrio harveyi</td><td>JF930417.1</td></tr><tr><td>69</td><td>Vibrio harveyi</td><td>JF930435.1</td></tr></tbody></table>

Appendix B. List of *mreB* sequences of *V. harveyi* and *V. harveyi*-related species, aligned in order to identify divergent regions.

<table><thead><tr><td>No.</td><td>Species</td><td>GenBank accession no.</td></tr></thead><tbody><tr><td>1</td><td>Vibrio harveyi</td><td>JN033881.1</td></tr><tr><td>2</td><td>Vibrio campbellii</td><td>HQ449952.1</td></tr><tr><td>3</td><td>Vibrio campbellii</td><td>HQ449949.1</td></tr><tr><td>4</td><td>Vibrio parahaemolyticus</td><td>DQ907440.1</td></tr><tr><td>5</td><td>Vibrio rotiferianus</td><td>HQ540716.1</td></tr><tr><td>6</td><td>Vibrio alginolyticus</td><td>JQ015322.1</td></tr><tr><td>7</td><td>Vibrio owensii</td><td>HQ540729.1</td></tr><tr><td>8</td><td>Vibrio mimicus</td><td>DQ907430.1</td></tr><tr><td>9</td><td>Vibrio natriegens</td><td>JF930491.1</td></tr><tr><td>10</td><td>Vibrio communis</td><td>JF930439.1</td></tr></tbody></table>
Appendix C. Sanger sequencing results obtained from the *ftsZ*, *recA* and *gyrB* sequences after performing a BLAST analysis.

<table><thead><tr><th rowspan="2">Strain/<br>Isolate ID</th><th colspan="3">Genes</th><th colspan="3">recA</th><th colspan="3">gyrB</th></tr><tr><th>ftsZ</th><th>% ID</th><th>e-value</th><th>Description</th><th>% ID</th><th>e-value</th><th>Description</th><th>% ID</th><th>e-value</th></tr></thead><tbody><tr><td>LMG 19046</td><td>V. campbellii</td><td>99.84</td><td>0.0</td><td>V. campbellii</td><td>98.77</td><td>0.0</td><td>V. campbellii</td><td>96.09</td><td>0.0</td></tr><tr><td>LMG 19047</td><td>V. owensii</td><td>99.50</td><td>0.0</td><td>V. rotiferianus</td><td>98.92</td><td>0.0</td><td>V. owensii</td><td>99.62</td><td>0.0</td></tr><tr><td>Vh2</td><td>V. jasicida</td><td>99.68</td><td>0.0</td><td>V. jasicida</td><td>99.22</td><td>0.0</td><td>V. jasicida</td><td>100</td><td>0.0</td></tr><tr><td>Vh5</td><td>V. harveyi</td><td>99.85</td><td>0.0</td><td>V. harveyi</td><td>99.53</td><td>0.0</td><td>V. harveyi</td><td>99.54</td><td>0.0</td></tr><tr><td>Vh8</td><td>V. harveyi</td><td>100</td><td>0.0</td><td>V. harveyi</td><td>99.40</td><td>0.0</td><td>V. harveyi</td><td>100</td><td>0.0</td></tr><tr><td>Vh15</td><td>V. rotiferianus</td><td>99.68</td><td>0.0</td><td>V. rotiferianus</td><td>99.54</td><td>0.0</td><td>V. rotiferianus</td><td>99.38</td><td>0.0</td></tr><tr><td>Vh22</td><td>V. harveyi</td><td>100</td><td>0.0</td><td>V. harveyi</td><td>99.40</td><td>0.0</td><td>V. harveyi</td><td>100</td><td>0.0</td></tr><tr><td>Vh25</td><td>V. harveyi</td><td>100</td><td>0.0</td><td>V. harveyi</td><td>99.39</td><td>0.0</td><td>V. harveyi</td><td>99.88</td><td>0.0</td></tr><tr><td>Vh28</td><td>V. owensii</td><td>99.68</td><td>0.0</td><td>V. owensii</td><td>99.07</td><td>0.0</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>Vh29</td><td>V. harveyi</td><td>100</td><td>0.0</td><td>V. harveyi</td><td>99.55</td><td>0.0</td><td>V. harveyi</td><td>99.84</td><td>0.0</td></tr><tr><td>Vt1</td><td>V. splendidus</td><td>99.84</td><td>0.0</td><td>V. atlanticus</td><td>100</td><td>0.0</td><td>V. tasmaniensis</td><td>98.00</td><td>0.0</td></tr><tr><td>Vt2</td><td>V. splendidus</td><td>99.70</td><td>0.0</td><td>V. atlanticus</td><td>98.45</td><td>0.0</td><td>V. tasmaniensis</td><td>98.36</td><td>0.0</td></tr><tr><td>Vg1</td><td>V. gigantis</td><td>99.83</td><td>0.0</td><td>ND</td><td>ND</td><td>ND</td><td>V. crassostreae</td><td>98.83</td><td>0.0</td></tr><tr><td>Vp1</td><td>V. rotiferianus</td><td>99.82</td><td>0.0</td><td>V. rotiferianus</td><td>98.60</td><td>0.0</td><td>V. rotiferianus</td><td>99.28</td><td>0.0</td></tr></tbody></table>

% ID, percent identity

ND, not detected (no PCR product was obtained)
Appendix D. Rooted phylogenic reconstruction based on whole genome sequences of LMG 19047 collection strain, Vt1 and Vg1 isolates and genome sequences of *Vibrio* spp. available on NCBI. Distance tree was generated with neighbor joining algorithm.

<figure><img src="image_36.png" /></figure>
# Schéma de synthèse

**Objectif** : Quantifier spécifiquement l'espèce *V. harveyi* à la fois dans des échantillons d'eau et de biofilms d'origine aquacole

## 1. Développement de la PCR en temps réel spécifique à V. harveyi

<figure><img src="image_37.png" /></figure>

## 2. Adaptation de la méthode de PCR en temps réel pour le traitement d'échantillons d'eau et de biofilms d'origine aquacole

<figure><img src="image_38.png" /></figure>

**Conclusion** : La PCR en temps réel mise au point en ciblant le gène *mreB* permet de quantifier *V. harveyi* directement dans les échantillons d'eau et de biofilms d'origine aquacole. Celle-ci sera par la suite utilisée dans le **Chapitre IV** afin de suivre l'évolution spatiale et temporelle de *V. harveyi* sur Aquanord.

Figure 26 : Schéma de synthèse du Chapitre II, relatif au développement d'une PCR en temps réel pour la détection et la quantification de *Vibrio harveyi* en milieu aquacole.
<figure><img src="image_39.png" /></figure>
# Chapitre III.

Développement de la Luvibase, une base de données permettant l'identification par MALDI-TOF MS des espèces du clade Harveyi

## Sommaire

<table><tr><td>1. Contexte</td><td>118</td></tr><tr><td>2. Utilisation de la base de données Bruker et de la VibrioBase</td><td>119</td></tr><tr><td>3. Développement et validation de la Luvibase : Article 2</td><td>122</td></tr><tr><td>Article 2</td><td>123</td></tr><tr><td>Abstract</td><td>123</td></tr><tr><td>1. Introduction</td><td>124</td></tr><tr><td>2. Materials and methods</td><td>126</td></tr><tr><td>3. Results</td><td>130</td></tr><tr><td>4. Discussion</td><td>138</td></tr><tr><td>5. Conclusion</td><td>141</td></tr><tr><td>References</td><td>143</td></tr><tr><td>Supplemental data</td><td>145</td></tr><tr><td>Schéma de synthèse</td><td>146</td></tr></table>
# 1. Contexte

À ce jour, la méthode de référence la plus couramment employée pour l'identification bactérienne est le séquençage du gène codant pour la sous-unité 16S de l'ARN ribosomal (ARNr). Ce gène cible universel a l'avantage d'être présent dans le génome de toutes les bactéries, présentant des séquences hautement conservées et des séquences spécifiques à chaque espèce. Toutefois, ces séquences étant très conservées au sein de certaines espèces de *Vibrio* et notamment des espèces du clade Harveyi, cette méthode ne permet pas une identification fiable au niveau de l'espèce (Gomez-Gil et al., 2004). De plus, celle-ci est coûteuse, fastidieuse et difficilement applicable au quotidien. Ainsi, pour établir un diagnostic rapide, de nombreux laboratoires vétérinaires se sont tournés vers la spectrométrie de masse de type matrix-assisted laser desorption ionization – time of flight (MALDI-TOF MS). Plusieurs études ont déjà démontré la fiabilité de cette technique pour l'identification des espèces appartenant au genre *Vibrio* (Hazen et al., 2009, Dieckmann et al., 2010, Eddabra et al., 2012, Schirmeister et al., 2014, Erler et al., 2015). L'analyse par MALDI-TOF MS, des protéines exprimées par les bactéries, génère des valeurs de rapports masse sur charge (m/z) spécifiques. Ces valeurs permettent de générer un spectre de masse, incluant des signaux de masse spécifiques (Clark et al., 2013). L'identification repose ensuite sur la comparaison de ces spectres de masse avec les spectres enregistrés dans une base de données. Ainsi, la fiabilité de l'identification dépend fortement des spectres déposés dans la base de données. À ce jour, l'une des bases de données commerciales la plus utilisée est la base de données Bruker BioTyper, majoritairement générée pour le diagnostic clinique et manquant de spectres de souches d'origine environnementale ou aquacole. Il est toutefois possible d'élaborer des bases de données, dites sur-mesure, afin d'améliorer l'identification d'espèces d'intérêt. Par exemple, Erler et al. (2015) ont mis au point la VibrioBase, une base de données permettant d'améliorer l'identification d'espèces du genre *Vibrio*.

L'objectif de ce chapitre consiste à relater le développement d'une méthode permettant d'identifier les espèces du clade Harveyi, de manière fiable, par MALDI-TOF MS. Pour cela, la base de données Bruker BioTyper ainsi que la VibrioBase, mise au point par Erler et al. (2015),
ont d'abord été testées, afin d'identifier des isolats d'origine aquacole. Ces tests aboutissant à des erreurs d'identification, la « Luvibase », une base de données sur-mesure permettant d'identifier les espèces appartenant au clade Harveyi a été élaborée. Cette base de données a été valorisée par une publication dans la revue en *Open Access, Frontiers in Microbiology*, la rendant ainsi disponible pour tous (Mougin et al., 2020b).

## 2. Utilisation de la base de données Bruker et de la VibrioBase

Dans un premier temps, les spectres de masse des 85 souches et isolats précédemment identifiés dans le *Chapitre II, Article 1* ont été acquis par MALDI-TOF MS en utilisant la technique préconisée par Bruker et détaillée dans l'**Article 2**. Les spectres obtenus ont ensuite été comparés à la base de données Bruker BioTyper ver. 9.0.0.0, puis la base de données Bruker BioTyper ver. 9.0.0.0 implémentée avec la VibrioBase (Erler et al., 2015). Les résultats obtenus sont disponibles dans le Tableau 7.

<table><caption>Tableau 7 : Comparaison entre les résultats d'identification obtenus par MALDI-TOF MS en utilisant la base de données Bruker BioTyper ver. 9.0.0.0. et les résultats obtenus en utilisant la base de données Bruker BioTyper, implémentée avec la VibrioBase. Les 85 isolats utilisés ont été précédemment identifiés dans le *Chapitre II, Article 1*. Remplissage vert = identification identique à l'espèce attribuée, remplissage rouge = identification différente de l'espèce attribuée</caption><thead><tr><th rowspan="3">Souches/Isolats<br>ID</th><th rowspan="3">Espèce attribuée</th><th colspan="4">Identification par MALDI-TOF MS</th></tr><tr><th colspan="2">Base de données Bruker</th><th colspan="2">Base de données Bruker + VibrioBase</th></tr><tr><th>Identification</th><th>log-score</th><th>Identification</th><th>log-score</th></tr></thead><tbody><tr><td colspan="6">Souches de collection incluses dans la Luvibase (23)</td></tr><tr><td>LMG 23688</td><td>V. harveyi</td><td>V. harveyi</td><td>● 1,91</td><td>V. harveyi/V. campbellii</td><td>● 1,91</td></tr><tr><td>LMG 18299</td><td>V. harveyi</td><td>V. harveyi</td><td>● 2,28</td><td>V. harveyi/V. campbellii</td><td>● 2,28</td></tr><tr><td>LMG 22894</td><td>V. harveyi</td><td>V. harveyi</td><td>● 2,36</td><td>V. harveyi/V. campbellii</td><td>● 2,36</td></tr><tr><td>LMG 22895</td><td>V. harveyi</td><td>V. harveyi</td><td>● 2,22</td><td>V. harveyi/V. campbellii</td><td>● 2,22</td></tr><tr><td>LMG 11755</td><td>V. harveyi</td><td>V. harveyi</td><td>● 2,33</td><td>V. harveyi/V. campbellii</td><td>● 2,36</td></tr><tr><td>LMG 21363</td><td>V. campbellii</td><td>V. parahaemolyticus</td><td>● 2,09</td><td>V. harveyi/V. campbellii</td><td>● 2,15</td></tr><tr><td>LMG 11256</td><td>V. campbellii</td><td>V. parahaemolyticus</td><td>● 2,17</td><td>V. parahaemolyticus</td><td>● 2,17</td></tr><tr><td>CIP 70.67</td><td>V. campbellii</td><td>V. harveyi</td><td>● 2,20</td><td>V. harveyi/V. campbellii</td><td>● 2,23</td></tr><tr><td>LMG 16862</td><td>V. campbellii</td><td>V. harveyi</td><td>● 2,22</td><td>V. harveyi/V. campbellii</td><td>● 2,23</td></tr><tr><td>LMG 16863</td><td>V. campbellii</td><td>V. harveyi</td><td>● 2,25</td><td>V. harveyi/V. campbellii</td><td>● 2,30</td></tr><tr><td>DSMZ 23055<sup>T</sup></td><td>V. owensii</td><td>V. harveyi</td><td>● 2,14</td><td>V. harveyi/V. campbellii</td><td>● 2,31</td></tr><tr><td>LMG 25430<sup>T</sup></td><td>V. owensii</td><td>V. harveyi</td><td>● 2,16</td><td>V. harveyi/V. campbellii</td><td>● 2,28</td></tr><tr><td>LMG 20370</td><td>V. owensii</td><td>V. harveyi</td><td>● 2,15</td><td>V. harveyi/V. campbellii</td><td>● 2,35</td></tr><tr><td>LMG 4043</td><td>V. owensii</td><td>V. harveyi</td><td>● 2,27</td><td>V. harveyi/V. campbellii</td><td>● 2,37</td></tr><tr><td>LMG 21456</td><td>V. rotiferianus</td><td>V. rotiferianus</td><td>● 2,28</td><td>V. harveyi/V. campbellii</td><td>● 2,29</td></tr></tbody></table>
<table><thead><tr><th rowspan="3">Souches/Isolats<br>ID</th><th rowspan="3">Espèce attribuée</th><th colspan="4">Identification par MALDI-TOF MS</th></tr><tr><th colspan="2">Base de données Bruker</th><th colspan="2">Base de données Bruker + VibrioBase</th></tr><tr><th>Identification</th><th>log-score</th><th>Identification</th><th>log-score</th></tr></thead><tbody><tr><td>LMG 2850<sup>T</sup></td><td>V. parahaemolyticus</td><td>V. parahaemolyticus</td><td>● 2,40</td><td>V. parahaemolyticus</td><td>● 2,54</td></tr><tr><td>LMG 16838</td><td>V. parahaemolyticus</td><td>V. parahaemolyticus</td><td>● 2,39</td><td>V. parahaemolyticus</td><td>● 2,54</td></tr><tr><td>LMG 11650</td><td>V. alginolyticus</td><td>V. alginolyticus</td><td>● 2,24</td><td>V. alginolyticus</td><td>● 2,44</td></tr><tr><td>LMG 4409<sup>T</sup></td><td>V. alginolyticus</td><td>V. alginolyticus</td><td>● 2,12</td><td>V. alginolyticus</td><td>● 2,28</td></tr><tr><td>LMG 10950</td><td>V. natriegens</td><td>V. natriegens</td><td>● 2,16</td><td>V. natriegens</td><td>● 2,19</td></tr><tr><td>LMG 25266<sup>T</sup></td><td>V. azureus</td><td>V. parahaemolyticus</td><td>● 2,17</td><td>V. parahaemolyticus</td><td>● 2,17</td></tr><tr><td>CECT 8524</td><td>V. jasicida</td><td>V. harveyi</td><td>● 2,41</td><td>V. harveyi/V. campbellii</td><td>● 2,42</td></tr><tr><td>CECT 7298<sup>T</sup></td><td>V. sinaloensis</td><td>V. brasiliensis</td><td>● 1,87</td><td>V. xuii</td><td>● 1,88</td></tr><tr><th colspan="6">Souches de collection incluses dans la base de données Bruker</th></tr><tr><td>LMG 4044<sup>T</sup></td><td>V. harveyi</td><td>V. harveyi</td><td>● 2,18</td><td>V. harveyi/V. campbellii</td><td>● 2,18</td></tr><tr><td>LMG 7890</td><td>V. harveyi</td><td>V. harveyi</td><td>● 2,28</td><td>V. harveyi/V. campbellii</td><td>● 2,28</td></tr><tr><td>LMG 19643</td><td>V. harveyi</td><td>V. harveyi</td><td>● 2,15</td><td>V. harveyi/V. campbellii</td><td>● 2,15</td></tr><tr><td>LMG 11216</td><td>V. campbellii</td><td>V. harveyi</td><td>● 2,25</td><td>V. harveyi/V. campbellii</td><td>● 2,26</td></tr><tr><td>DSMZ 17186<sup>T</sup></td><td>V. rotiferianus</td><td>V. rotiferianus</td><td>● 2,19</td><td>V. harveyi/V. campbellii</td><td>● 2,27</td></tr><tr><td>DSMZ 19137<sup>T</sup></td><td>V. mytili</td><td>V. mytili</td><td>● 2,21</td><td>V. mytili</td><td>● 2,21</td></tr><tr><th colspan="6">Autres souches de collection testées</th></tr><tr><td>LMG 23442</td><td>V. harveyi</td><td>V. harveyi</td><td>● 2,30</td><td>V. harveyi/V. campbellii</td><td>● 2,27</td></tr><tr><td>LMG 16832</td><td>V. harveyi</td><td>V. harveyi</td><td>● 2,35</td><td>V. harveyi/V. campbellii</td><td>● 2,35</td></tr><tr><td>LMG 23678</td><td>V. harveyi</td><td>V. harveyi</td><td>● 2,36</td><td>V. harveyi/V. campbellii</td><td>● 2,36</td></tr><tr><td>LMG 11226</td><td>V. harveyi</td><td>V. harveyi</td><td>● 2,28</td><td>V. harveyi/V. campbellii</td><td>● 2,28</td></tr><tr><td>LMG 23680</td><td>V. harveyi</td><td>V. harveyi</td><td>● 2,36</td><td>V. harveyi/V. campbellii</td><td>● 2,36</td></tr><tr><td>LMG 10946</td><td>V. campbellii</td><td>V. parahaemolyticus</td><td>● 2,19</td><td>V. parahaemolyticus</td><td>● 2,19</td></tr><tr><td>LMG 10947</td><td>V. owensii</td><td>V. harveyi</td><td>● 2,26</td><td>V. harveyi/V. campbellii</td><td>● 2,34</td></tr><tr><td>LMG 11659</td><td>V. owensii</td><td>V. harveyi</td><td>● 2,21</td><td>V. harveyi/V. campbellii</td><td>● 2,34</td></tr><tr><td>CECT 526<sup>T</sup></td><td>V. natriegens</td><td>V. natriegens</td><td>● 2,27</td><td>V. natriegens</td><td>● 2,37</td></tr><tr><th colspan="6">Isolats</th></tr><tr><td>Vh1, 3, 4, 5, 6, 7,<br>8, 9, 10, 11, 12,<br>13, 14, 16, 17, 18,<br>19, 20, 21, 22, 23,<br>24, 25, 26, 29, 31,<br>32, 33, 34, 35, 36,<br>37, 38, 43, 45, 47,<br>48, 51, 52, 53</td><td>V. harveyi (40)</td><td>V. harveyi</td><td>● 2,19*</td><td>V. harveyi/V. campbellii</td><td>● 2,05*</td></tr><tr><td>Vh2</td><td>V. jasicida</td><td>V. harveyi</td><td>● 2,18</td><td>V. harveyi/V. campbellii</td><td>● 2,18</td></tr><tr><td>Vh15</td><td>V. rotiferianus</td><td>V. rotiferianus</td><td>● 2,29</td><td>V. rotiferianus</td><td>● 2,29</td></tr><tr><td>Vh28</td><td>V. owensii</td><td>V. harveyi</td><td>● 2,00</td><td>V. harveyi/V. campbellii</td><td>● 2,18</td></tr><tr><td>Vt1, 2</td><td>V. tasmaniensis (2)</td><td>V. tasmaniensis</td><td>● 2,02*</td><td>V. tasmaniensis</td><td>● 2,19*</td></tr><tr><td>Vg1</td><td>V. gigantis</td><td>V. gigantis</td><td>● 2,15</td><td>V. harveyi/V. campbellii</td><td>● 2,18</td></tr><tr><td>Vp1</td><td>V. rotiferianus</td><td>V. parahaemolyticus</td><td>● 2,16</td><td>V. rotiferianus</td><td>● 2,16</td></tr></tbody></table>

Un log-score (i) supérieur à 2,3 indique une identification hautement probable à l'espèce (disque vert foncé), (ii) un log-score entre 2,0 et 2,3 une identification probable à l'espèce (disque vert clair), (iii) un log-score compris entre 1,7 et 2,0 indique une identification probable au genre seulement (disque jaune), et (iv) un log-score inférieur à 1,7 indique aucune similarité significative entre un MSP de référence de la base de données et le spectre MALDI-TOF MS expérimental.

<footnote>* Moyenne de log-score</footnote>
D'après ces résultats, la base de données Bruker BioTyper ver. 9.0.0.0 a généré une identification correcte à l'espèce pour seulement 75,3 % des souches et isolats. L'utilisation de la base de données Bruker BioTyper ver. 9.0.0.0 implémentée avec la VibrioBase a, quant à elle, généré une identification correcte à l'espèce pour 77,6 %. Néanmoins, lors du développement de la VibrioBase, les spectres de référence des espèces *Vibrio harveyi* et *Vibrio campbellii* ont été enregistrés au sein d'un même groupe *V. harveyi/campbellii*, rendant ces deux espèces indissociables. En effet, la méthode utilisée, afin d'identifier les isolats environnementaux avant implémentation dans la base de données, c'est-à-dire le séquençage du gène *rpoB*, ne permettait pas de différencier ces deux espèces, génétiquement très proches (Erler *et al.*, 2015). Ainsi cette base de données n'est pas adaptée à l'identification et à la différenciation des espèces potentiellement pathogènes retrouvées en ferme aquacole, et notamment sur Aquanord-Ichtus.

C'est dans ce contexte qu'une nouvelle base de données, la Luvibase, a été mise au point.
## 3. Développement et validation de la Luvibase : Article 2

**Statut de l'article** Publié dans *Frontiers in Microbiology* (IF : 4,235) : Mougin *et al*. (2020b)

**Objectif de l'étude** L'objectif était de développer une méthode afin d'identifier les espèces du clade Harveyi, de manière fiable, par MALDI-TOF MS.

**Résultats principaux** Une base de données, nommée Luvibase, a été élaborée avec 23 souches de collection, précédemment présentées dans le *Chapitre II, Article 1*, appartenant aux espèces *V. alginolyticus*, *V. azureus*, *V. campbellii*, *V. harveyi*, *V. jasicida*, *V. natriegens*, *V. owensii*, *V. parahaemolyticus*, *V. rotiferianus* et *V. sinaloensis*. Le séquençage du gène codant pour l'ARNr 16S de ces 23 souches a démontré un manque de pouvoir de résolution, par rapport à la technologie MALDI-TOF MS. Enfin, la Luvibase a été validée grâce à l'identification correcte, au niveau de l'espèce, de 62 souches et isolats précédemment identifiés dans le *Chapitre II, Article 1*.

**Conclusion** L'implémentation de la base de données Bruker BioTyper avec la Luvibase permet l'identification fiable au niveau de l'espèce des souches et isolats appartenant au clade Harveyi. Cette base de données permet ainsi d'améliorer l'identification, au niveau de l'espèce, d'isolats d'origine aquacole.
## Article 2

# Rapid identification of *Vibrio* species of the Harveyi clade using MALDI-TOF MS profiling with main spectral profile database implemented with an in-house database: Luvibase

Julia Mougin<fnref n="1" />, Christophe Flahaut<fnref n="1" />, Roxane Roquigny<fnref n="1" />, Maryse Bonnin-Jusserand<fnref n="1" />, Thierry Grard<fnref n="1" /> and Cédric Le Bris<fnref n="1" />

**Keywords:** MALDI-TOF MS, *Vibrio*, Harveyi clade, in-house database, Luvibase, environmental isolates

## Abstract

*Vibrio* bacteria, and particularly members of the Harveyi clade, are the causative agents of vibriosis. This disease is responsible for mass mortality events and important economic losses on aquaculture farms. Improvements in surveillance and diagnosis are needed to successfully manage vibriosis outbreaks. 16S rRNA gene sequencing is generally considered to be the gold standard for bacterial identification but the cost and long processing time make it difficult to apply for routine identification. In contrast, MALDI-TOF MS offers rapid diagnosis and is commonly used in veterinary laboratories today. The major limiting factor for using this technique is the low environmental bacterial diversity in the commonly available databases. Here, we demonstrate that the sole use of the commercially available Bruker BioTyper database is not fully adequate for identifying *Vibrio* bacteria isolated from aquaculture farms. We therefore developed a new

<footnote n="1">¹ Univ. Littoral Côte d'Opale, UMR 1158 BioEcoAgro, Institut Charles Viollette, USC ANSES, INRAe, Univ. Artois, Univ. Lille, Univ. Picardie Jules Verne, Univ. Liège, Yncréa, F-62200 Boulogne-sur-Mer, France</footnote>
in-house database named Luvibase, composed of 23 reference MALDI-TOF mass spectra profiles obtained from *Vibrio* collection strains, mostly belonging to the Harveyi clade. The comparison of the accuracy of MALDI-TOF MS profiling and 16S rRNA gene sequencing revealed a lack of resolution for 16S rRNA gene sequencing. In contrast, MALDI-TOF MS profiling proved to be a more reliable tool for resolving species-level variations within the Harveyi clade. Finally, combining the Luvibase with the Bruker ver. 9.0.0.0 database, led to successful identification of 47 *Vibrio* isolates obtained from moribund abalone, seabass and oysters. Thus, the use of Luvibase allow for increased confidence in identifying *Vibrio* species belonging to the Harveyi clade.

# 1. Introduction

Bacteria belonging to the *Vibrio* genus, are highly abundant in aquatic environments (Thompson *et al.*, 2004a). Some of them are well-known enteric human pathogens such as *Vibrio parahaemolyticus*, *V. vulnificus* or even *V. cholerae*, known as the causative agent of pandemic cholera (Bonnin-Jusserand *et al.*, 2017). Other *Vibrio* species are pathogenic to aquatic animals such as several species belonging to the Harveyi clade. Associated with a great variety of crustaceans, mollusks or fishes, these bacteria are particularly problematic in aquaculture, causing vibriosis outbreaks and high economic losses (Austin *et Zhang*, 2006, Novriadi, 2016, Ina-Salwany *et al.*, 2019). The health risk to aquatic animals and the potential development of zoonosis heighten the need for rapid and reliable identification of *Vibrio* species belonging to the Harveyi clade (Cantas *et Suer*, 2014, Destoumieux-Garzón *et al.*, 2018). Thus, in case of vibriosis outbreaks, appropriate control measures could be designed and rapidly applied. The gold standard method for species identification is 16S rRNA gene sequencing (Chatterjee *et Haldar*, 2012). However, several studies have highlighted the poor discriminatory power of 16S rRNA sequence-based bacterial identification of *Vibrio* species, due to high genome similarity (Gomez-Gil *et al.*, 2004, Hernandez *et Olmos*, 2004). Furthermore, this technique is often cost-ineffective and time-consuming, making it labor-intensive to apply in aquaculture facilities in a context of vibriosis outbreaks.
Thus, for rapid diagnosis, most veterinary laboratories routinely use matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI TOF MS). The key benefits of this technique are the high throughput and speed combined with automation that provide rapid sample processing on a large scale (Fujinami et al., 2011, Singhal et al., 2015). Previous studies have demonstrated that MALDI-TOF MS is a valuable tool for discriminating among Vibrio spp. and closely related species (Hazen et al., 2009, Dieckmann et al., 2010, Eddabra et al., 2012, Schirmeister et al., 2014, Erler et al., 2015). The identification of bacteria relies on the comparison of experimental MALDI TOF MS profiles with reference MALDI TOF MS profiles recorded in a database. The most commonly and widely used database, is the commercially available Bruker BioTyper database (Bruker France S.A.S, Wissembourg, Germany). This latter is mainly intended for human clinical diagnosis. Although considerable efforts have recently been made to implement reference MALDI-TOF MS profiles from environmental strains, this database still lacks coverage of environmental bacterial isolates, particularly marine bacteria (Emami et al., 2012). Therefore, the use of this database for environmental surveys is limited.

To overcome this limitation, one suitable solution is to create an in-house database, adapted to the identification of the species of interest. For instance, Erler et al. (2015) created an available free in-house database, named VibrioBase, in order to improve the identification of Vibrio isolates at the species level. This database was mainly compiled using Vibrio environmental isolates potentially pathogenic for human and thus lacks spectra from potential aquatic animal pathogens. In particular, species belonging to the Harveyi clade, such as V. owensii, V. rotiferianus, V. azureus, or V. jasicida are missing from this database. Moreover, spectra from V. harveyi and V. campbellii have been included in a common V. harveyi/campbellii group, making their discrimination impossible. Previous studies have already highlighted the need to find accurate tools allowing discrimination between these two species (Gomez-Gil et al., 2004, Ruwandeepika et al., 2010, Cano-Gomez et al., 2011). The impact of V. campbellii in aquaculture facilities may indeed have been underestimated due to unreliable identification.
The aim of this study was to improve the identification of *Vibrio* belonging to the Harveyi clade to the species level using MALDI-TOF MS profiling. More specifically, our objective was to develop a method for the accurate identification of pathogens obtained from moribund abalone (*Haliotis tuberculata*), seabass (*Dicentrarchus labrax*) and oyster (*Crassostrea gigas*) from aquaculture farms. These strains have been previously identified using PCR and sequencing by Mougin *et al.* (2020a). Given that the MALDI BioTyper was not adequate for species identification, we constructed an in-house database, named Luvibase. Reference MALDI-TOF mass spectra profiles, also shortened as mass spectra profiles (MSPs), obtained from 23 *Vibrio* collection strains were incorporated into the Luvibase. We then compared MALDI-TOF MS profiling with 16S rRNA gene sequencing for species identification. Finally, the identification of 62 environmental isolates was performed using the Bruker BioTyper ver. 9.0.0.0 database supplemented with the Luvibase, to provide evidence of the usefulness of this augmented database.

## 2. Materials and methods

### 2.1. Bacterial strains and culture conditions

A total of 85 strains were used in this study (Table 1). These strains were described and identified by PCR, sequencing of house-keeping genes or whole genome sequencing in a previous study (Mougin *et al.*, 2020a). Briefly, (i) 38 collection strains were obtained from the Belgian Coordinated Collections of Microorganisms (BCCM/LMG), the German Collection of Microorganisms and Cell Cultures (DSMZ), the Spanish Type Culture Collection (CECT), and the Institut Pasteur Collection (CIP), (ii) 35 isolates were obtained from a fish farm (Aquanord-Ichtus, Gravelines, France) raising seabass (*Dicentrarchus labrax*) and (iii) 12 isolates were isolated by the French Research Institute for the Exploitation of the Sea (IFREMER) from oysters (*Crassostrea gigas*) and abalone (*Haliotis tuberculata*). Before all experiments, bacteria were first sub-cultured on marine agar (MA), (Difco Laboratories, Detroit, MI, USA) and incubated for 24 h, at growth temperature. Second, a single colony forming unit (CFU) was plated on MA (Difco Laboratories) and incubated 24 h, at growth temperature, to ensure the
purity of the isolates. This growth temperature was 37 °C, except for LMG 11216T, Vh2, Vh3, Vt1, Vt2 and Vg1, for which the growth temperature was 25 °C.

## 2.2. 16S rRNA gene sequencing and sequence analysis

The DNA of 23 *Vibrio* collection strains was extracted from pure culture, isolated from MA agar using the DNeasy Blood&Tissue kit (Qiagen, Hilden, Germany) and an automated nucleic acid extractor QIAcube Connect (Qiagen), according to the manufacturer's instructions. The final elution volume was 100 µL. The extracted DNA was then stored at -20 °C until use. PCR amplification of the 1465 bp 16S rRNA gene fragment (V1-V8 region) was performed as described in Bauer *et al.* (2018) using the primers 27f (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492r (5'-ACGGYTACCTTGTTACGACTT-3'). The primers were synthetized by TIB MOLBIOL (TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany) and were suspended in nuclease-free water to reach a final concentration of 10 µM and stored at -20 °C. The PCR reaction mixture contained 25 µL of 2× Platinum™ Green Hot Start PCR Master Mix (Invitrogen, Carlsbad, CA, USA), 1 µL of each primer (10 µM), 5 µL of template DNA, and 18 µL of nuclease-free water to a final volume of 50 µL. The PCR reaction was run on a Thermal Cycler (Applied Biosystems, Forster City, CA, USA), under the following conditions: 3 min at 95 °C, followed by 30 cycles of 30 s at 95 °C, 30 s at 55 °C, and 1 min at 72 °C. The final cycle was followed by an additional 7 min of extension at 72 °C. Each run included a no template control (NTC), DNA-free. The size of the PCR product was verified using ethidium bromide agarose gel electrophoresis (2%). The PCR products of expected size were then paired-end sequenced, using Sanger sequencing by GenoScreen (Lille, France).

Sequence analysis was performed using the nucleotide BLAST search program with the GenBank database (NCBI). A strict identification criterion of ≥ 99% sequence identity for identification to the species level was applied (Janda *et Abbott*, 2007). A distance tree was generated using CLC Genomics Workbench ver. 20.0.2. The unweighted pair grouping method with arithmetic-mean (UPGMA) hierarchical clustering algorithm was used with the Jukes-Cantor distance.
## 2.3. Preparation of bacterial extracts and target-loading for MALDI-TOF MS profiling

The cellular proteins of 23 *Vibrio* collection strains were extracted from bacterial cells according to manufacturer's recommendations (Bruker France S.A.S., Wissembourg, Germany). Fresh colonies were picked with a 1 µL inoculation loop and placed in 300 µL of water (ULC/MS-CC/SFC, Biosolve Chimie, Dieuze, France). A volume of 900 µL of pure ethanol (VWR International, Fontenay-sous-Bois, France) was added. The tube was vortexed for 1 min and centrifuged at 13000×g for 2 min. The supernatant was removed and the pellet was centrifuged a second time for 15 s, to eliminate as much ethanol as possible. Thereafter, pellets were air dried under a fume hood for 20 min. Once dried, pellets were dissolved into 30 µL of 70% formic acid (Alfa Aesar, Kandel, Germany). An equal volume of 100% acetonitrile (Biosolve Chimie) was added and the solution was mixed carefully by pipetting up and down. The tube was then centrifuged at 13000×g for 2 min. The extracts were stocked at -20 °C until use. Subsequently, 1 µL of the supernatant from each protein extract was spotted onto a clean ground steel MTP 384-target plate (Bruker France S.A.S.) in 24 replicates. After air-drying, each spot was overlaid with 1 µL of a 10 mg.mL⁻¹ matrix solution of α-cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile and 2.5% trifluoroacetic acid (Sigma-Aldrich, St. Louis, MO, USA).

## 2.4. Acquisition of MALDI-TOF MS profiles

MALDI-TOF-MS analysis was performed using an Autoflex SpeedTM (Bruker France S.A.S.), running Flexcontrol ver. 3.4 software (Bruker France S.A.S.). The Bruker bacterial test standard (BTS, Bruker France S.A.S.) was used to calibrate the mass spectrometer before each run, according to the manufacturer's recommendations. Mass spectra were acquired in the positive linear ion mode across a mass-to-charge (m/z) ratio of 2000-20000 Da. The mass spectra were acquired using the manufacturer's automatic method MBT_FC.par. For each MALDI-TOF MS profile, mass spectra obtained from 5000 laser shots in 1000 shot steps performed randomly on different areas of the spot were accumulated.
## 2.5. Creation of the Luvibase, an in-house main spectral profile database

Each of the 23 MALDI-TOF MS profiles obtained from 24 MALDI-target spots were analyzed and processed using FlexAnalysis ver. 3.4 (Bruker France S.A.S.). MALDI-TOF MS profiles with a signal intensity lower than $10^4$ arbitrary units (a.u.) were discarded. A minimum of 18 MALDI-TOF MS profiles were used to create an MSP using MALDI BioTyper Compass Explorer ver. 4.1 software (Bruker France S.A.S.). All 23 MSPs were registered in an in-house database called Luvibase. In order to ensure the repeatability of the assay, tree cultures of each strain were performed and the similarity of mass spectra of each culture was confirmed.

An MSP dendrogram was generated using MALDI BioTyper Compass Explorer ver. 4.1 (Bruker France S.A.S.) using the Euclidean distance measure and a complete coverage algorithm.

## 2.6. Bacterial identification using MALDI-TOF MS profiling

A single CFU, isolated from a fresh bacterial culture, was spotted onto a clean ground steel MTP 384-target plate (Bruker France S.A.S.) in four replicates. Then, 1 µL of 70% formic acid (Alfa Aesar) was overlaid on each spot. After air-drying at room temperature, 1 µL of CHCA solution in 50% acetonitrile and 2.5% trifluoroacetic acid (Sigma-Aldrich) was added to each spot and air-dried. Subsequently, the above mentioned MALDI-TOF parameters were used to acquire the MALDI-TOF MS profiles.

Each MALDI-TOF MS profile was first compared to the MSPs of the Bruker BioTyper database ver. 9.0.0.0 (Bruker France S.A.S.) and then to the MSPs of the Bruker BioTyper database ver. 9.0.0.0 supplemented with the Luvibase. The concordance degree between experimental MALDI-TOF MS profiles and the MSPs was evaluated using the log-score calculated by MALDI BioTyper Compass Explorer ver. 4.1 (Bruker France S.A.S.). A log-score greater than 2.3 indicates a highly probable identification at the species level. A log-score between 2.0 and 2.3 indicates a probable identification at the species level. A log-score between 1.7 and 2.0 indicates a probable identification at the genus level only. A log-score below 1.7 indicates no significant similarities between MSPs included in the databases and the tested profile.
# 3. Results

## 3.1. Identification of *Vibrio* bacteria using the Bruker ver. 9.0.0.0 database

We acquired MALDI-TOF MS profiles for 85 *Vibrio* bacterial strains, classified into 13 *Vibrio* species mostly belonging to the Harveyi clade, were acquired. These spectra were first compared to the MSPs included in the Bruker ver. 9.0.0.0 database. The bacterial identifications previously obtained in the study of Mougin *et al.* (2020a) were then compared with the MALDI-TOF MS profile-based bacterial identifications. The matching rates were 100% at the genus level and only 75.3% at the species level, assuming a log-score of at least 2.0 required for probable species identification (Table 1). A total of 19 bacterial strains were misidentified using the Bruker ver. 9.0.0.0 database, involving the 6 following species: *V. campbellii*, *V. owensii*, *V. azureus*, *V. jasicida*, *V. sinaloensis*, and *V. rotiferianus*. (i) All *V. campbellii* collection strains, LMG 21363, LMG 11256, CIP 70.67, LMG 16862, LMG 16863, LMG 11216 and LMG 10946 were misidentified as *V. parahaemolyticus* or *V. harveyi* (log-score > 2); (ii) all *V. owensii* collections strains, DSMZ 23055<sup>T</sup>, LMG 25430<sup>T</sup>, LMG 20370, LMG 4043, LMG 11659, LMG 10947 and the *V. owensii* isolate Vh28 were misidentified as *V. harveyi* (log-score > 2); (iii) the *V. jasicida* collection strain CECT 8524 and the *V. jasicida* isolate Vh2 were misidentified as *V. harveyi* (log-score > 2); (iv) the *V. azureus* collection strains LMG 25266<sup>T</sup> was misidentified as *V. parahaemolyticus* (log-score > 2); (v) the *V. rotiferianus* isolate Vp1 was misidentified as *V. parahaemolyticus* whereas the *V. rotiferianus* collection strain LMG 21456 was correctly identified (log-score > 2) and (vi) the *V. sinaloensis* collection strain CECT 7298<sup>T</sup> was identified to the genus level only (1.7 < log-score < 2). Therefore, we constructed an in-house database to provide a reliable identification of bacteria belonging to these 6 species.
<table><caption>Table 1. Comparison between 16S rRNA gene sequencing identification results and MALDI-TOF MS profile-based identification of Vibrio species using the Bruker BioTyper ver. 9.0.0.0 database alone and supplemented with the Luvibase. Bacterial isolates were previously identified using PCR and sequencing by Mougin et al. (2020a). Green shading = MALDI-TOF MS identifications matching the assigned species identifications; red shading = MALDI-TOF MS identifications not matching the assigned species identifications.</caption><thead><tr><th rowspan="3">Tested strains/Isolates</th><th rowspan="3">Assigned species</th><th colspan="2">16S rRNA gene sequencing</th><th colspan="4">MALDI-TOF MS</th></tr><tr><th rowspan="2">Identification</th><th rowspan="2">Percent similarity (%)</th><th colspan="2">Bruker database</th><th colspan="2">Bruker database + Luvibase</th></tr><tr><th>Identification</th><th>log-score</th><th>Identification</th><th>log-score</th></tr></thead><tbody><tr><td colspan="8">Collection strains in the Luvibase (23)</td></tr><tr><td>LMG 23688</td><td>V. harveyi</td><td>V. harveyi</td><td>99</td><td>V. harveyi</td><td>● 1.91</td><td>V. harveyi</td><td>● 2.64</td></tr><tr><td>LMG 18299</td><td>V. harveyi</td><td>V. harveyi</td><td>100</td><td>V. harveyi</td><td>● 2.28</td><td>V. harveyi</td><td>● 2.36</td></tr><tr><td>LMG 22894</td><td>V. harveyi</td><td>V. harveyi</td><td>100</td><td>V. harveyi</td><td>● 2.36</td><td>V. harveyi</td><td>● 2.45</td></tr><tr><td>LMG 22895</td><td>V. harveyi</td><td>V. harveyi</td><td>100</td><td>V. harveyi</td><td>● 2.22</td><td>V. harveyi</td><td>● 2.45</td></tr><tr><td>LMG 11755</td><td>V. harveyi</td><td>V. harveyi</td><td>100</td><td>V. harveyi</td><td>● 2.33</td><td>V. harveyi</td><td>● 2.44</td></tr><tr><td>LMG 21363</td><td>V. campbellii</td><td>V. campbellii/V. harveyi</td><td>100</td><td>V. parahaemolyticus</td><td>● 2.09</td><td>V. campbellii</td><td>● 2.38</td></tr><tr><td>LMG 11256</td><td>V. campbellii</td><td>V. campbellii/V. harveyi</td><td>100</td><td>V. parahaemolyticus</td><td>● 2.17</td><td>V. campbellii</td><td>● 2.32</td></tr><tr><td>CIP 70.67</td><td>V. campbellii</td><td>V. campbellii/V. harveyi</td><td>99</td><td>V. harveyi</td><td>● 2.20</td><td>V. campbellii</td><td>● 2.44</td></tr><tr><td>LMG 16862</td><td>V. campbellii</td><td>V. campbellii/V. harveyi</td><td>100</td><td>V. harveyi</td><td>● 2.22</td><td>V. campbellii</td><td>● 2.45</td></tr><tr><td>LMG 16863</td><td>V. campbellii</td><td>V. campbellii/V. harveyi</td><td>100</td><td>V. harveyi</td><td>● 2.25</td><td>V. campbellii</td><td>● 2.30</td></tr><tr><td>DSMZ 23055<sup>T</sup></td><td>V. owensii</td><td>V. owensii/V. hyugaensis/V. vulnificus/V. harveyi</td><td>100</td><td>V. harveyi</td><td>● 2.14</td><td>V. owensii</td><td>● 2.37</td></tr><tr><td>LMG 25430<sup>T</sup></td><td>V. owensii</td><td>V. hyugaensis/V. owensii/V. vulnificus/V. harveyi</td><td>100</td><td>V. harveyi</td><td>● 2.16</td><td>V. owensii</td><td>● 2.35</td></tr><tr><td>LMG 20370</td><td>V. owensii</td><td>V. hyugaensis/V. owensii/V. harveyi</td><td>100</td><td>V. harveyi</td><td>● 2.15</td><td>V. owensii</td><td>● 2.27</td></tr><tr><td>LMG 4043</td><td>V. owensii</td><td>V. vulnificus/V. hyugaensis/V. owensii/V. harveyi</td><td>100</td><td>V. harveyi</td><td>● 2.27</td><td>V. owensii</td><td>● 2.37</td></tr><tr><td>LMG 21456</td><td>V. rotiferianus</td><td>V. harveyi/V. campbellii</td><td>100</td><td>V. rotiferianus</td><td>● 2.28</td><td>V. rotiferianus</td><td>● 2.44</td></tr><tr><td>LMG 2850<sup>T</sup></td><td>V. parahaemolyticus</td><td>V. parahaemolyticus</td><td>100</td><td>V. parahaemolyticus</td><td>● 2.40</td><td>V. parahaemolyticus</td><td>● 2.29</td></tr><tr><td>LMG 16838</td><td>V. parahaemolyticus</td><td>V. parahaemolyticus/V. mediterranei/V. natriegens/V. neonatus/V. aureus/V. alginolyticus</td><td>100</td><td>V. parahaemolyticus</td><td>● 2.39</td><td>V. parahaemolyticus</td><td>● 2.42</td></tr><tr><td>LMG 11650</td><td>V. alginolyticus</td><td>V. alginolyticus/V. natriegens/V. aureus</td><td>100</td><td>V. alginolyticus</td><td>● 2.24</td><td>V. alginolyticus</td><td>● 2.33</td></tr><tr><td>LMG 4409<sup>T</sup></td><td>V. alginolyticus</td><td>V. alginolyticus/V. aureus</td><td>100</td><td>V. alginolyticus</td><td>● 2.12</td><td>V. alginolyticus</td><td>● 2.13</td></tr><tr><td>LMG 10950</td><td>V. natriegens</td><td>V. natriegens</td><td>97.18</td><td>V. natriegens</td><td>● 2.16</td><td>V. natriegens</td><td>● 2.11</td></tr></tbody></table>
<table><thead><tr><th rowspan="3">Tested strains/Isolates</th><th rowspan="3">Assigned species</th><th rowspan="3">16S rRNA gene sequencing</th><th rowspan="3">Percent similarity (%)</th><th colspan="4">MALDI-TOF MS</th></tr><tr><th colspan="2">Bruker database</th><th colspan="2">Bruker database + Luvibase</th></tr><tr><th>Identification</th><th>log-score</th><th>Identification</th><th>log-score</th></tr></thead><tbody><tr><td>LMG 25266<sup>T</sup></td><td>V. <i>azureus</i></td><td>V. <i>azureus</i>/V. <i>campbellii</i>/<br/>V. <i>alginolyticus</i></td><td>100</td><td>V. <i>parahaemolyticus</i></td><td>● 2.17</td><td>V. <i>azureus</i></td><td>● 2.35</td></tr><tr><td>CECT 8524</td><td>V. <i>jasicida</i></td><td>V. <i>azureus</i>/V. <i>hyugaensis</i></td><td>100</td><td>V. <i>harveyi</i></td><td>● 2.41</td><td>V. <i>jasicida</i></td><td>● 2.54</td></tr><tr><td>CECT 7298<sup>T</sup></td><td>V. <i>sinaloensis</i></td><td>V. <i>sinaloensis</i></td><td>100</td><td>V. <i>brasiliensis</i></td><td>● 1.87</td><td>V. <i>sinaloensis</i></td><td>● 2.36</td></tr><tr><td colspan="8">Collection strains in the Bruker database tested (6)</td></tr><tr><td>LMG 4044<sup>T</sup></td><td>V. <i>harveyi</i></td><td></td><td></td><td>V. <i>harveyi</i></td><td>● 2.18</td><td>V. <i>harveyi</i></td><td>● 2.24</td></tr><tr><td>LMG 7890</td><td>V. <i>harveyi</i></td><td></td><td></td><td>V. <i>harveyi</i></td><td>● 2.28</td><td>V. <i>harveyi</i></td><td>● 2.46</td></tr><tr><td>LMG 19643</td><td>V. <i>harveyi</i></td><td></td><td></td><td>V. <i>harveyi</i></td><td>● 2.15</td><td>V. <i>harveyi</i></td><td>● 2.22</td></tr><tr><td>LMG 11216</td><td>V. <i>campbellii</i></td><td></td><td></td><td>V. <i>harveyi</i></td><td>● 2.25</td><td>V. <i>campbellii</i></td><td>● 2.44</td></tr><tr><td>DSMZ 17186<sup>T</sup></td><td>V. <i>rotiferianus</i></td><td></td><td></td><td>V. <i>rotiferianus</i></td><td>● 2.19</td><td>V. <i>rotiferianus</i></td><td>● 2.42</td></tr><tr><td>DSMZ 19137<sup>T</sup></td><td>V. <i>mytili</i></td><td></td><td></td><td>V. <i>mytili</i></td><td>● 2.21</td><td>V. <i>mytili</i></td><td>● 2.00</td></tr><tr><td colspan="8">Other collection strains tested (9)</td></tr><tr><td>LMG 23442</td><td>V. <i>harveyi</i></td><td></td><td></td><td>V. <i>harveyi</i></td><td>● 2.30</td><td>V. <i>harveyi</i></td><td>● 2.31</td></tr><tr><td>LMG 16832</td><td>V. <i>harveyi</i></td><td></td><td></td><td>V. <i>harveyi</i></td><td>● 2.35</td><td>V. <i>harveyi</i></td><td>● 2.43</td></tr><tr><td>LMG 23678</td><td>V. <i>harveyi</i></td><td></td><td></td><td>V. <i>harveyi</i></td><td>● 2.36</td><td>V. <i>harveyi</i></td><td>● 2.33</td></tr><tr><td>LMG 11226</td><td>V. <i>harveyi</i></td><td></td><td></td><td>V. <i>harveyi</i></td><td>● 2.28</td><td>V. <i>harveyi</i></td><td>● 2.31</td></tr><tr><td>LMG 23680</td><td>V. <i>harveyi</i></td><td></td><td></td><td>V. <i>harveyi</i></td><td>● 2.36</td><td>V. <i>harveyi</i></td><td>● 2.39</td></tr><tr><td>LMG 10946</td><td>V. <i>campbellii</i></td><td></td><td></td><td>V. <i>parahaemolyticus</i></td><td>● 2.19</td><td>V. <i>campbellii</i></td><td>● 2.37</td></tr><tr><td>LMG 10947</td><td>V. <i>owensii</i></td><td></td><td></td><td>V. <i>harveyi</i></td><td>● 2.26</td><td>V. <i>owensii</i></td><td>● 2.33</td></tr><tr><td>LMG 11659</td><td>V. <i>owensii</i></td><td></td><td></td><td>V. <i>harveyi</i></td><td>● 2.21</td><td>V. <i>owensii</i></td><td>● 2.32</td></tr><tr><td>CECT 526<sup>T</sup></td><td>V. <i>natriegens</i></td><td></td><td></td><td>V. <i>natriegens</i></td><td>● 2.27</td><td>V. <i>natriegens</i></td><td>● 2.25</td></tr><tr><td colspan="8">Identified isolates tested (47)</td></tr><tr><td>Vh1, 3, 4, 5, 6,<br/>7, 8, 9, 10, 11,<br/>12, 13, 14, 16,<br/>17, 18, 19, 20,<br/>21, 22, 23, 24,<br/>25, 26, 29, 31,<br/>32, 33, 34, 35,</td><td>V. <i>harveyi</i> (40)</td><td></td><td></td><td>V. <i>harveyi</i></td><td>● 2.19*</td><td>V. <i>harveyi</i></td><td>● 2.28*</td></tr></tbody></table>
<table><thead><tr><th rowspan="3">Tested strains/Isolates</th><th rowspan="3">Assigned species</th><th rowspan="3">16S rRNA gene sequencing</th><th rowspan="3">Percent similarity (%)</th><th colspan="4">MALDI-TOF MS</th></tr><tr><th colspan="2">Bruker database</th><th colspan="2">Bruker database + Luvibase</th></tr><tr><th>Identification</th><th>log-score</th><th>Identification</th><th>log-score</th></tr></thead><tbody><tr><td>36, 37, 38, 43, 45, 47, 48, 51, 52, 53</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Vh2</td><td>V. jasicida</td><td></td><td></td><td>V. harveyi</td><td>2.18</td><td>V. jasicida</td><td>2.39</td></tr><tr><td>Vh15</td><td>V. rotiferianus</td><td></td><td></td><td>V. rotiferianus</td><td>2.29</td><td>V. rotiferianus</td><td>2.38</td></tr><tr><td>Vh28</td><td>V. owensii</td><td></td><td></td><td>V. harveyi</td><td>2.00</td><td>V. owensii</td><td>2.29</td></tr><tr><td>Vt1, 2</td><td>V. tasmaniensis (2)</td><td></td><td></td><td>V. tasmaniensis</td><td>2.02*</td><td>V. tasmaniensis</td><td>2.02*</td></tr><tr><td>Vg1</td><td>V. gigantis</td><td></td><td></td><td>V. gigantis</td><td>2.15</td><td>V. gigantis</td><td>2.15</td></tr><tr><td>Vp1</td><td>V. rotiferianus</td><td></td><td></td><td>V. parahaemolyticus</td><td>2.16</td><td>V. rotiferianus</td><td>2.31</td></tr></tbody></table>

*Mean of log-scores

A log-score (i) greater than 2.3 indicates a highly probable identification at the species level (dark green circle), (ii) between 2.0 and 2.3 indicates a probable identification at the species level (light green circle), (iii) between 1.7 and 2.0 indicates a probable identification at the genus level only (yellow circle) and (iv) below 1.7 indicates no significant similarities between the reference MSPs included in the databases and the experimental MALDI-TOF MS profiles.
## 3.2. Creation of the in-house database, Luvibase

MSPs, derived from 23 *Vibrio* collection strains, were created and recorded in the in-house database: Luvibase (Tables 1 & 2). The Luvibase includes the MSPs of 12 collection strains classified into the 6 species that yielded unreliable identifications: *V. campbellii*, *V. owensii*, *V. azureus*, *V. jasicida*, *V. sinaloensis*, and *V. rotiferianus*. In addition, the MSPs of 11 collection strains classified as *V. harveyi*, *V. parahaemolyticus*, *V. alginolyticus* and *V. natriegens* were included in the Luvibase.

<table><caption>Table 2. Number of *Vibrio* strains included in the Bruker BioTyper database ver. 9.0.0.0 and in the Luvibase</caption><thead><tr><th>Species</th><th>No. of strains in the Bruker database</th><th>No. of strains in the Luvibase</th></tr></thead><tbody><tr><td>Vibrio harveyi</td><td>7</td><td>5</td></tr><tr><td>Vibrio campbellii</td><td>1</td><td>5</td></tr><tr><td>Vibrio owensii</td><td>0</td><td>4</td></tr><tr><td>Vibrio alginolyticus</td><td>5</td><td>2</td></tr><tr><td>Vibrio rotiferianus</td><td>1</td><td>1</td></tr><tr><td>Vibrio parahaemolyticus</td><td>9</td><td>2</td></tr><tr><td>Vibrio natriegens</td><td>2</td><td>1</td></tr><tr><td>Vibrio azureus</td><td>0</td><td>1</td></tr><tr><td>Vibrio jasicida</td><td>0</td><td>1</td></tr><tr><td>Vibrio sinaloensis</td><td>0</td><td>1</td></tr></tbody></table>

As illustrated in Figure 1, each MSP of the 10 *Vibrio* species implemented in the Luvibase, gave a unique and reproducible species-specific MSP. Minor visual differences were perceptible between these MSPs: they were clearly similar but not identical.
<figure><img src="image_40.png" /><figcaption>Figure 1. Main spectral profiles (MSPs) of the 23 *Vibrio* strains included in the Luvibase. The whole 2000-14000 m/z spectra are shown (a.u., arbitrary units; m/z, mass to charge ratio).</figcaption></figure>
### 3.3. Identification of the strains included in the Luvibase using 16S rRNA gene sequencing

The 16S rRNA gene sequences (V1-V8 region) of the 23 strains included in the Luvibase were obtained and identification was performed using the nucleotide BLAST search program with the GenBank database (NCBI). For most of the fragments, the sequence similarity obtained was above the strict threshold defined for identification at the species level (≥ 99% sequence similarity). The sequence similarity obtained from fragment acquired from the LMG 10950 *V. natriegens* collection strain was below the threshold (97.48%), and was thus identified at the genus level only. Nevertheless, 15/22 strains could not be identified due to high sequence similarities with more than one species (Table 1): strains belonging to *V. campbellii*, *V. owensii*, *V. rotiferianus*, *V. alginolyticus*, *V. azureus*, and *V. jasicida* could not be correctly identified. On both *V. parahaemolyticus* strains, only the LMG 2850T collection strain was correctly identified whereas the LMG 16838 collection strain was incorrectly identified. Strains belonging to *V. harveyi* and *V. sinaloensis* were correctly identified.

### 3.4. Comparison of 16S rRNA gene sequencing to MALDI-TOF profiling for identification of species included in the Luvibase

To evaluate the agreement between species discrimination using 16S rRNA gene sequencing and MALDI-TOF MS profiling, a hierarchical distance tree, based on the 16S rRNA gene sequences obtained from the 23 strains in the Luvibase, and a MSP dendrogram based on the MSPs included in the Luvibase were generated (Fig 2).
<figure><img src="image_41.png" /><figcaption>Figure 2. Comparison of (a) main spectral profile (MSP) dendrogram based on MALDI-TOF MS profiling analysis and (b) 16S rRNA gene-based distance tree resulting based on 16S rRNA gene sequencing of 23 *Vibrio* strains included in the Luvibase.</figcaption></figure>

Using both techniques, all strains belonging to the Harveyi clade were grouped together and *V. sinaloensis*, which belongs to the Orientalis clade, was easily distinguishable. Nevertheless, comparison between the MSP dendrogram and the 16S rRNA gene-based distance tree revealed that the clusters within the Harveyi clade differed between the two techniques. Analysis of clusters resulting from MALDI-TOF MS profiling highlighted species-specific groups, but analysis of clusters from 16S rRNA gene sequencing were not all species-specific. The 16S rRNA gene sequences of *V. alginolyticus*, *V. natriegens* and *V. parahaemolyticus* clustered together. These findings suggest that the two techniques have different taxonomic resolution.

**3.5. Identification of *Vibrio* bacteria using the Bruker ver. 9.0.0.0 database supplemented with the Luvibase**

To evaluate the effectiveness of the Luvibase for identification of unknown isolates, the 85 *Vibrio* bacteria were re-identified. The Bruker BioTyper database supplemented with the Luvibase and
bacterial identification results are summarized in Table 1. The matching rates were 100% at both the genus and the species levels, assuming a log-score of at least 2.0 required for probable species identification (Table 1).

Overall, the mean of log-score obtained with the Bruker BioTyper ver. 9.0.0.0 database was 2.21 ± 0.12, whereas the mean of log-score obtained with the Bruker BioTyper ver. 9.0.0.0 database supplemented with the Luvibase was 2.34 ± 0.12. Thus, the Bruker BioTyper ver. 9.0.0.0 database gave mostly “probable species identifications” (log-score < 2.3) while the augmented database gave “secure species identifications” (log-score ≥ 2.3).

All experiments were validated by negative and positive control spots. The negative control spots yielded no peaks or faint profiles that could not be identified by the system, and the positive control spots yielded the expected *Escherichia coli* DH5α identification score of 2.0-2.5.

# 4. Discussion

Genomic-based methods for *Vibrio* identification, such as PCR and sequencing, offer accurate identification, assuming the use of a specific and reliable target gene (Cano-Gomez *et al.*, 2009). However, they are also labor-intensive, requiring highly trained operators and are not particularly suitable for proceeding a large quantity of samples. On the contrary, MALDI-TOF MS is an automated, rapid, cost-effective and useful tool for bacterial species identification. This technique has been successfully used to discriminate closely related *Vibrio* species (Dieckmann *et al.*, 2010, Erler *et al.*, 2015). The major limitation is the lack of MSP diversity in the main available spectral profile databases (Clark *et al.*, 2013).

The Bruker BioTyper ver. 9.0.0.0 database includes 111 MSPs obtained from 53 different *Vibrio* species (Table S1). As expected, strains belonging to *V. owensii*, *V. azureus*, *V. jasicida* and *V. sinaloensis* species were not correctly identified because their MSPs are not included. Consistently, the *V. sinaloensis* spectrum did not specifically match any MSP and was identified to the genus level only. However, the MALDI-TOF MS profiles for *V. owensii*, *V. azureus* and
V. jasicida matched the MSPs of other Vibrio strains with log scores ≥ 2 or ≥ 2.3, the recommended standard thresholds for probable or secure species identification, respectively. These inaccurate bacterial identification matches highlight the high similarity of MALDI-TOF MS profiles between Vibrio species. In contrast, the Bruker BioTyper ver. 9.0.0.0 database does include one MSP for V. campbellii and V. rotiferianus. All MALDI-TOF MS profiles from strains classified as V. campbellii matched the MSPs of other Vibrio species. The MALDI-TOF MS profile from a V. rotiferianus isolate did not match the MSP of its own species. Nonetheless, the MALDI-TOF MS profiles from the other V. rotiferianus strains correctly matched the species MSP. These findings suggest that including only one MSP in a MSP database is not sufficient for secure species identification, perhaps partly due to the high heterogeneity between environmental isolates. For instance, the geographic diversity of V. parahaemolyticus isolates can lead to divergences in MALDI-TOF MS profiles (Hazen et al., 2009). To ensure high epidemiological coverage, more MSPs must be recorded in the bacterial identification databases. Thus, the reliable identification of the causative agents of disease outbreaks in aquaculture requires the construction of a database including different strains of V. parahaemolyticus and related species.

Here, we constructed a new in-house database, named Luvibase, in order to improve the identification of *Vibrio* belonging to the Harveyi clade at the species level. The MSPs of 23 *Vibrio* species were acquired and included. Overall, the use of the Luvibase in addition to the Bruker BioTyper ver. 9.0.0.0 database provided more reliable identification than the sole use of the Bruker BioTyper ver. 9.0.0.0 database. Moreover, the cluster analysis highlighted the capacity of MALDI-TOF MS profiling to discriminate between closely related species. Remarkably, the method allowed accurate identification and discrimination between *V. harveyi*, *V. campbellii* and *V. rotiferianus* whose 16S rRNA gene sequences have more than 99% similarity (Gomez-Gil et al., 2003, Gomez-Gil et al., 2004).

16S rRNA gene sequencing is currently considered the gold standard for bacterial identification. However, in our study, only a few strains could be correctly identified to the species level due to
high 16S rRNA gene sequence similarities between *Vibrio* species belonging to the Harveyi clade. Moreover, the 16S rRNA gene-based distance tree analysis highlighted a close relationship between the 16S rRNA gene sequences of *V. alginolyticus*, *V. natriegens* and *V. parahaemolyticus*. Importantly, the discrimination between these species proved to be laborious, because they shared high similarity in their 16S rRNA gene sequences (Dorsch et al., 1992, Thompson et al., 2009). Instead of the 16S rRNA gene sequencing, Dieckmann et al. (2010) demonstrated that the partial *rpoB* gene sequencing can resolve species-level variations between *V. alginolyticus* and *V. parahaemolyticus*. Thus, the 16S rRNA gene is not a reliable target gene for discrimination between these species. Generally speaking, 16S rRNA gene sequencing is not suitable for species identification within the Harveyi clade which form a tight cluster with more than 99% sequence similarities (Cano-Gomez et al., 2011). In contrast, MALDI-TOF MS profiling had higher resolving power. This technique is based on the analysis of a large spectrum of peptides/proteins generated from the whole cell, while 16S rRNA gene sequencing is based only on a 1500 bp gene. Thus, MALDI-TOF MS profiling enables the characterization of proteomic differences between bacteria with highly similar 16S rRNA gene sequences. For this reason, in our study, MALDI-TOF MS profiling gave a more accurate bacterial identification than 16S rRNA gene sequencing. In addition, there is no universal consensus of an established threshold value for sequence similarity above which strains can unambiguously be identified to the species level, using 16S rRNA gene sequencing (Janda et Abbott, 2007). Attempts to propose an optimal threshold of 98.65% when using 16S rRNA gene sequencing for species identification are stymied by the fact that some species share more than 99% gene sequence similarity (Kim et al., 2014a).

In contrast, the log-score threshold values for MALDI-TOF MS identification are well defined and universally applicable. Overall, the implementation of the Bruker BioTyper database along with the Luvibase, for bacterial identification using MALDI-TOF MS appears to be an appropriate, convenient and powerful tool to discriminate between closely related species belonging to the Harveyi clade.

Standard conditions must be well defined and applied to ensure reliability and reproducibility of MALDI-TOF MS identification. The divergence between methods can lead to divergence between
mass signals and thus MALDI-TOF MS profiles (Santos et al., 2016). Several studies have highlighted the importance of cell growth conditions, culture age, matrices, solvents and method of sample application on the target plate (Evason et al., 2001, Williams et al., 2003, Vargha et al., 2006). These parameters may affect the spectra and, thus the associated identifications. However, there is a controversy regarding the impact of growth conditions. Some studies claimed that even if the spectrum is slightly modified, identification to the species level remain possible (Valentine et al., 2005, Usbeck et al., 2013). For instance, the use of different culture media for *Yersinia enterocolitica*, *Bacillus subtilis*, and *Escherichia coli* identification by MALDI-TOF MS profiling showed an unambiguous core set of proteins, ensuring reliable identification regardless the culture media (Valentine et al., 2005). Recently, the identification of *V. anguillarum* pathogens isolated from seabass and seabream (*Sparus aurata*) revealed that differences in culture media and incubation period have no effect on MALDI-TOF MS profiles but divergences in growth temperature may affect MALDI-TOF MS profiles (Kazazić et al., 2019). Importantly, because the spectra of different species may not be affected by the same parameters, the effect of altering procedures should be studied for each single species. To our knowledge, no such studies have been undertaken for *Vibrio* species belonging to the Harveyi clade. Procedures clearly need to be standardized to ensure consistent identification between laboratories.

**5. Conclusion**

This work revealed discrepancies between *Vibrio* species discrimination using 16S rRNA gene sequencing and MALDI-TOF MS profiling. In our study, MALDI-TOF MS profiling provided better resolution because this technique is based on the analysis of a large spectrum of peptides/proteins from the whole cell, whereas 16S rRNA gene sequencing is based on a single 1500 bp gene. Therefore, 16S rRNA gene sequences exhibit high similarities between *Vibrio* species. However, the success of bacterial identification using MALDI-TOF profiling relies on the reference strains registered in databases. As demonstrated in the present study, the sole use of the Bruker BioTyper ver. 9.0.0.0 database is not suitable for identification of *Vibrio* isolates from aquaculture farms. For that reason, we constructed the Luvibase, an in-house database suitable
for the discrimination of Vibrio species belonging to the Harveyi clade. The Luvibase combined
with the Bruker BioTyper database can be used for pathogen identification in aquaculture farms.
The combination of these both databases led to reliable identification of the strains belonging to
V. campbellii, V. owensii, V. azureus, V. jasicida, V. sinaloensis, and V. rotiferianus, whereas
the use of the Bruker BioTyper database alone gave incorrect identifications. Therefore,
veterinary laboratories can use the augmented database for the rapid and reliable diagnosis of
vibriosis outbreaks. In the case of an identified outbreak, suitable control methods could be then
undertaken to limit the spread of the disease. The inclusion of additional environmental isolates
in the Luvibase could then expand the coverage and the use of this technique.

Funding

Julia Mougin would like to thank the Hauts-de-France regional council and ULCO for their
financial support of her PhD work. This work was funded by the French government, IFREMER
and the region Hauts-de-France as part of the CPER 2014-2020 MARCO project. This work was
also supported by a grant from FEAMP (PFEA470017FA1000006) as part of the LUVIBAR
project.

Acknowledgements

The authors thank Aquanord-Ichtus for contributing to the LUVIBAR project, especially
Guillaume Tielie, Anabelle Duhamel and Céline Doyen. The authors are also grateful to the
REALCAT platform funded by a French governmental grant administered by the French
National Research Agency (ANR) as part of the frame of the ‘Investments for the Future’
program (ANR-11-EQPX-0037) and Egon Heuson for helpful assistance. The authors thank the
National Reference Laboratory for mollusk diseases for sharing V. harveyi strains, and the French
Directorate General for Food (DGAL) for funding the NRL. Finally, the authors thank the
LABOCEA veterinary laboratory for sharing V. harveyi strains isolated from Aquanord-Ichtus.
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Supplemental data

<table><caption>Table S1. Species list and number of Vibrio strains included in the Bruker BioTyper database ver. 9.0.0.0.</caption><thead><tr><th>Species</th><th>No. of strains in the Bruker database</th></tr></thead><tbody><tr><td>Vibrio aerogenes</td><td>1</td></tr><tr><td>Vibrio aestuarianus</td><td>4</td></tr><tr><td>Vibrio agarivorans</td><td>1</td></tr><tr><td>Vibrio albensis</td><td>1</td></tr><tr><td>Vibrio alginolyticus</td><td>5</td></tr><tr><td>Vibrio anguillarum</td><td>7</td></tr><tr><td>Vibrio brasiliensis</td><td>1</td></tr><tr><td>Vibrio campbellii</td><td>1</td></tr><tr><td>Vibrio chagasii</td><td>1</td></tr><tr><td>Vibrio cincinnatiensis</td><td>1</td></tr><tr><td>Vibrio coralliilyticus</td><td>1</td></tr><tr><td>Vibrio cyclitrophicus</td><td>1</td></tr><tr><td>Vibrio diazotrophicus</td><td>6</td></tr><tr><td>Vibrio ezurae</td><td>1</td></tr><tr><td>Vibrio fluvialis</td><td>3</td></tr><tr><td>Vibrio fortis</td><td>1</td></tr><tr><td>Vibrio furnissii</td><td>3</td></tr><tr><td>Vibrio gazogenes</td><td>1</td></tr><tr><td>Vibrio gigantis</td><td>1</td></tr><tr><td>Vibrio harveyi</td><td>7</td></tr><tr><td>Vibrio hispanicus</td><td>1</td></tr><tr><td>Vibrio ichthyoenteri</td><td>2</td></tr><tr><td>Vibrio kanaloae</td><td>1</td></tr><tr><td>Vibrio lentus</td><td>1</td></tr><tr><td>Vibrio mediterranei</td><td>2</td></tr><tr><td>Vibrio metschnikovii</td><td>2</td></tr><tr><td>Vibrio mimicus</td><td>1</td></tr><tr><td>Vibrio mytili</td><td>1</td></tr><tr><td>Vibrio natriegens</td><td>2</td></tr><tr><td>Vibrio navarrensis</td><td>5</td></tr><tr><td>Vibrio neptunius</td><td>1</td></tr><tr><td>Vibrio nereis</td><td>1</td></tr><tr><td>Vibrio nigripulchritudo</td><td>1</td></tr><tr><td>Vibrio ordalii</td><td>1</td></tr><tr><td>Vibrio orientalis</td><td>1</td></tr><tr><td>Vibrio ostreicida</td><td>3</td></tr><tr><td>Vibrio pacinii</td><td>1</td></tr><tr><td>Vibrio parahaemolyticus</td><td>9</td></tr><tr><td>Vibrio pectenicida</td><td>2</td></tr><tr><td>Vibrio pelagius</td><td>1</td></tr><tr><td>Vibrio penaeicida</td><td>1</td></tr><tr><td>Vibrio pomeroui</td><td>1</td></tr><tr><td>Vibrio ponticus</td><td>1</td></tr><tr><td>Vibrio proteolyticus</td><td>1</td></tr><tr><td>Vibrio rotiferianus</td><td>1</td></tr><tr><td>Vibrio ruber</td><td>1</td></tr><tr><td>Vibrio rumoiensis</td><td>1</td></tr><tr><td>Vibrio scophthalmi</td><td>2</td></tr><tr><td>Vibrio splendidus</td><td>1</td></tr><tr><td>Vibrio superstes</td><td>1</td></tr><tr><td>Vibrio tasmaniensis</td><td>1</td></tr><tr><td>Vibrio vulnificus</td><td>11</td></tr><tr><td>Vibrio xuii</td><td>1</td></tr><tr><td>TOTAL</td><td>111</td></tr></tbody></table>
# Schéma de synthèse

**Objectif** : Identifier les espèces appartenant au clade Harveyi de manière fiable par MALDI-TOF MS

<figure><img src="image_42.png" /></figure>

**Conclusion** : La Luvibase, implémentée dans la base de données Bruker BioTyper, permet l'identification, fiable au niveau de l'espèce, des espèces appartenant au clade Harveyi. Cette base de données permet d'améliorer l'identification des isolats d'origine aquacole et plus particulièrement isolés d'Aquanord-Ichtus.

Figure 27 : Schéma de synthèse du Chapitre III, relatif au développement de la Luvibase, une base de données permettant l'identification par MALDI-TOF MS des espèces du clade Harveyi.
# Chapitre IV.

## Distribution spatiale et temporelle des
Vibrionaceae et de l'espèce *Vibrio harveyi*
dans un élevage aquacole de bars communs

### Sommaire

1. Contexte .................................................................................................................. 148

1.1. Aquanord-Ichtus en quelques chiffres................................................................ 148

1.2. Le site d'élevage.................................................................................................. 148

1.3. La problématique de la vibriose sur Aquanord-Ichtus........................................ 149

2. Distribution spatiale et temporelle des *Vibrio* en milieu aquacole : Article 3 ........................................ 151

Article 3......................................................................................................................... 153

Abstract......................................................................................................................... 153

1. Introduction............................................................................................................ 154

2. Material and methods.......................................................................................... 156

3. Results.................................................................................................................. 162

4. Discussion............................................................................................................ 170

5. Conclusion............................................................................................................ 174

References.................................................................................................................. 175

Supplemental data.................................................................................................... 178

3. Etude préliminaire des capacités d'adhésion et de formation de biofilms ........................................ 180

3.1. Tentative d'étude *in vitro* de formation de biofilms sur béton........................................ 180

3.2. Quantification relative de la formation de biofilm par coloration au cristal violet .... 181

3.3. Caractérisation du biofilm grâce au Biofilm Ring Test® .................................................. 184

Schéma de synthèse........................................................................................................ 190
# 1. Contexte

## 1.1. Aquanord-Ichtus en quelques chiffres

Aquanord-Ichtus est une entreprise spécialisée dans le secteur piscicole, leader français de l'élevage de bars communs (*Dicentrarchus labrax*) et de daurades royales (*Sparus aurata*). Cette ferme aquacole, fondée en 1982, fait partie du groupe Gloria Maris, promoteur d'une aquaculture marine durable et de qualité. Aquanord-Ichtus se situe à Gravelines (59) dans le Nord de la France, à proximité du site de la Centrale Nucléaire de Production d'Electricité (CNPE). Elle produit environ 1800 tonnes de poissons par an, avec un chiffre d'affaire de plus de 14 000 000 €. Cette ferme de grossissement, commercialise des bars de 300 g à 2,5 kg, âgés de 2 à 4 ans avec les labels Agriculture Biologique (AB) et *Friend of the Sea*, et les certifications Origine France Garantie et Charte Qualité-Aquaculture de nos régions®. Les alevins proviennent de l'Ecloserie marine de Gravelines Ichtus située à proximité (Gloria Maris Groupe, 2020).

## 1.2. Le site d'élevage

Aquanord-Ichtus est une exploitation de type intensive, semi-fermée pratiquant l'élevage en bassins en terre. Afin de maintenir la qualité de l'eau d'élevage (salinité, pH, évacuation des excréments...) ainsi qu'une température optimale pour la croissance du bar commun (idéalement 20 °C – 24 °C (Vázquez et Muñoz-Cueto, 2014)), les bassins sont alimentés par deux circuits d'eau de mer : une arrivée d'eau, dite froide, provenant de l'eau de mer environnante et une arrivée d'eau, dite chaude, provenant du système de refroidissement des réacteurs de la CNPE. L'eau des bassins est un mélange contrôlé de ces deux arrivées d'eau. L'hiver, la proportion d'eau provenant du canal d'eau chaude sera plus importante qu'en été, période au cours de laquelle les bassins sont uniquement alimentés par le canal d'eau froide. Ces informations sont résumées dans la Figure 28.
<figure><img src="image_43.png" /><figcaption>Figure 28 : Implantation et fonctionnement de la ferme aquacole Aquanord-Ichtus sur le site de Gravelines. A. Photographie aérienne de la ferme Aquanord-Ichtus (CNPE : Centrale Nucléaire de Production d'Electricité). B. Photographie des 3 bassins d'élevage utilisés lors de l'étude. C. Schéma du fonctionnement d'un module d'élevage sur Aquanord-Ichtus. Le canal d'alimentation est un mélange contrôlé d'eau chaude, provenant du canal de rejet de la CNPE (A.b.) et d'eau froide, provenant du canal d'amenée (A.a.), l'eau du canal d'évacuation est rejetée dans la mer du Nord.</figcaption></figure>

## 1.3. La problématique de la vibriose sur Aquanord-Ichtus

Les bactéries du genre Vibrio et plus particulièrement les espèces Vibrio anguillarum et Vibrio harveyi sont responsables d'épisodes récurrents de vibriose sur Aquanord-Ichtus lors des périodes estivales. Ces épisodes entraînent une mortalité massive des bars communs ainsi qu'une perte économique considérable. Le risque de déclenchement de vibriose, liée à V. anguillarum, est aujourd'hui globalement contrôlé grâce à l'administration d'un vaccin commercial par voix intrapéritonéale. En revanche, à ce jour, aucun vaccin permettant de lutter contre V. harveyi n'est disponible sur le marché. Pour pallier cette absence, Aquanord-Ichtus utilise un auto-vaccin administré par voix intrapéritonéale, mis au point à partir de souches de V. harveyi pathogènes, isolées à partir de rates de poissons moribonds, lors de précédents épisodes de vibriose. Cet auto-vaccin est renouvelé chaque année en fonction de l'occurrence de nouvelles souches pathogènes. Ces vaccins sont donc d'efficacité variable et il est fréquent d'observer un épisode de vibriose
lorsque les juvéniles sortent de couverture vaccinale, c'est-à-dire en général lorsqu'ils atteignent
environ plus de 200 g. De plus, les vaccins ne sont administrés que chez les individus de plus de
60 g du fait de la fragilité et de la difficulté de manipuler des poissons de petite taille. Les
individus de moins de 60 g et de plus de 200 g ne sont donc pas protégés contre la vibriose. En
cas de déclenchement d'un épisode, le débit d'eau dans les bassins est augmenté afin de renouveler
l'eau et de purifier l'environnement. En cas d'échec et en dernier recours, l'antibiotique
oxytetracycline est administré par voie orale, en l'intégrant dans les aliments.

Les bactéries du genre *Vibrio* sont ubiquitaires du milieu aquatique. Il n'est donc pas surprenant
de les retrouver sur Aquanord-Ichtus. Néanmoins, selon les données issues du plan de surveillance
de la CNPE, en 2018, les bactéries cultivables appartenant au genre *Vibrio* étaient 38 fois plus
abondantes au point de rejet (canal d'évacuation de la ferme aquacole + canal de rejet d'eau
chaude de la CNPE) par rapport au canal d'amenée (eau de mer issue de l'environnement naturel)
(Antajan et al., 2019). Cette différence peut, en partie, s'expliquer par la hausse de la température
de l'eau (+ 10 °C) traversant la CNPE, favorisant la multiplication des bactéries du genre *Vibrio*.
Ceci signifie que l'alimentation en eau chaude de la ferme aquacole, pourrait être une source
probable de bactéries potentiellement pathogènes pour le bar commun. Cette augmentation peut
aussi s'expliquer par l'activité de la ferme. Les bactéries pourraient se multiplier dans les bassins
d'élevage, riches en nutriments. Une autre hypothèse serait que les bactéries du genre *Vibrio* sont
des bactéries résidentes au sein de la ferme, sous forme de biofilms. Les bactéries incorporées
dans un biofilm sont plus résistantes aux flux environnementaux, antibiotiques et désinfectants.
Il a d'ailleurs été prouvé que l'espèce V. harveyi est capable de former des biofilms sur le béton
(Karunasagar et al., 1996). Les biofilms peuvent donc induire une persistance des souches
potentiellement pathogènes sur Aquanord-Ichtus. L'abondance de bactéries du genre *Vibrio*
potentiellement pathogènes dans le canal d'alimentation d'eau chaude, dans les bassins et dans
les biofilms formés sur les parois des bassins en béton pourraient donc induire un risque pour le
bar commun.
La gestion du risque lié à l'espèce *V. harveyi* et aux autres espèces potentiellement pathogènes, implique la compréhension de leur dynamique au sein de la ferme aquacole Aquanord-Ichtus. Pour cela, une étude quantitative est nécessaire afin de caractériser l'évolution spatiale et temporelle de leur abondance. Une étude qualitative est également indispensable afin d'identifier une évolution de la diversité des populations bactériennes et plus particulièrement des Vibrionaceae. Enfin, les causes de ces évolutions doivent ensuite être considérées au travers d'une étude des corrélations impliquant différents facteurs environnementaux biotiques et abiotiques.

## 2. Distribution spatiale et temporelle des *Vibrio* en milieu aquacole : *Article 3*

**Statut de l'article** Soumis dans *Aquaculture* (IF : 3,224)

**Objectif de l'étude** Evaluer le risque *Vibrio* au sein de la ferme aquacole Aquanord-Ichtus par l'étude de la distribution spatiale et temporelle des Vibrionaceae cultivables à 37 °C et plus particulièrement de *V. harveyi*, ainsi que par l'étude de l'impact de différents facteurs environnementaux biotiques et abiotiques.

**Démarche expérimentale** Une campagne d'échantillonnage a été entreprise d'avril à octobre 2018 sur Aquanord-Ichtus. L'étude portait sur les arrivées d'eau alimentant toute la ferme : l'eau froide provenant de l'environnement naturel et l'eau chaude provenant du système de refroidissement de la CNPE, ainsi que sur 3 bassins d'élevage identiques, faisant figure de triplicatas. Deux types d'échantillons ont été collectés en triplicatas comme détaillé dans le *Chapitre I* : (i) des échantillons d'eau provenant des arrivées d'eau et des bassins, collectés chaque semaine, (ii) ainsi que des échantillons de biofilms, collectés toute les deux semaines, provenant des bassins et plus particulièrement du dispositif d'échantillonnage mis au point dans le *Chapitre I* (pour cela 100 morceaux de béton ont été immergés dans chaque bassin). Un total de 609 échantillons a été collecté avec 465 échantillons d'eau et 144 échantillons de biofilms. Ces derniers ont été analysés grâce aux méthodes mises au point dans les *Chapitre II*, *Article 1* et
**Chapitre III, Article 2.** La PCR en temps réel a permis de déterminer l'abondance de *V. harveyi* dans chaque compartiment étudié au cours du temps. La technique de MALDI-TOF MS, ainsi que l'utilisation de la base de données Bruker BioTyper ver. 9.0.0.0 implémentée avec la Luvibase ont permis d'identifier les bactéries isolées sur TCBS à 37 °C, température de croissance de la plupart des espèces du clade Harveyi. Enfin, une étude des corrélations intégrant différents facteurs environnementaux a été réalisée afin d'identifier les éléments influencant les évolutions de l'abondance de *V. harveyi* et des Vibrionaceae cultivables à 37 °C.

**Résultats principaux** Des espèces potentiellement pathogènes pour le bar commun (*Photobacterium damselae*, *V. alginolyticus*, *V. harveyi*, *V. owensii* et *V. rotiferianus*) subsistent au sein de la ferme, pendant une grande partie de l'année, non seulement sous forme planctonique mais également sous forme de biofilms. Ces espèces sont présentes à la fois dans les arrivées d'eau et dans les bassins. L'analyse des corrélations, intégrant différents facteurs environnementaux, a permis de déceler une corrélation entre la température et l'abondance de *V. harveyi* ou plus généralement des Vibrionaceae cultivables à 37 °C, non seulement dans l'eau d'élevage mais également dans les biofilms.

**Conclusion** La présence du principal agent pathogène *V. harveyi* tout au long de l'année, au sein de la ferme aquacole représente un risque constant pour le bar commun. Le contrôle et l'éradication de cet agent s'avèrent potentiellement difficiles, à cause de la présence de *V. harveyi* sous forme de biofilms, pouvant conférer une résistance accrue aux antibiotiques et désinfectants, et induisant ainsi une persistance de souches potentiellement pathogènes dans les bassins. Le risque lié à *V. harveyi* pourrait augmenter au cours des années à venir, en raison d'une augmentation de la température de l'eau, liée au réchauffement climatique.
## Article 3

# Abundance and spatial patterns over time of Vibrionaceae and *Vibrio harveyi* in water and biofilm from a seabass aquaculture facility

Julia Mouginª, Roxane Roquignyª, Christophe Flahautª, Maryse Bonnin-Jusserandª, Thierry Grardª and Cédric Le Brisª

**Keywords:** Aquaculture, *Dicentrarchus labrax*, MALDI-TOF MS, *Vibrio harveyi*, Vibrionaceae, real-time PCR

## Abstract

One of the major threats to the seabass (*Dicentrarchus labrax*) aquaculture sector is the development of vibriosis, a disease caused mainly by bacteria from the family Vibrionaceae, especially *Vibrio harveyi*. This disease can result in important economic losses or severe public health problems. Furthermore, bacteria can form biofilms on the walls of aquaculture tanks, leading to the persistence of potentially pathogenic bacteria on fish farms. Given the relationship between abundance in the surrounding environment and prevalence in fish, monitoring the presence of *V. harveyi* in rearing water and biofilms over time may help better understand the dynamics of the pathogen and factors that increase the risk of vibriosis outbreaks.

In this study, the dynamics and spatial patterns of cultivable Vibrionaceae and *V. harveyi* in water and biofilms from rearing tanks and in the cold- and hot-water inlets supplying the farm were monitored for 7 months. Several potential seabass pathogens were isolated:

<footnote>ª Univ. Littoral Côte d'Opale, UMR 1158 BioEcoAgro, Institut Charles Viollette, USC ANSES, INRAe, Univ. Artois, Univ. Lille, Univ. Picardie Jules Verne, Univ. Liège, Yncréa, F-62200 Boulogne-sur-Mer, France</footnote>
*Photobacterium damselae*, *Vibrio alginolyticus*, *Vibrio harveyi*, *Vibrio owensii* and *Vibrio rotiferianus*. However, *V. harveyi* remained the most problematic pathogen. Its relatively constant presence in water and biofilm samples throughout the sampling campaign represents a risk for vibriosis outbreaks. Moreover, the correlation between water temperature and the abundance of *V. harveyi* suggests that the risk fluctuates throughout the year. Obtaining qualitative data regarding the relative diversity of viable culturable Vibrionaceae and quantitative data on the abundance of *V. harveyi* over time in a seabass aquaculture facility is the essential first step for designing new solutions to manage vibriosis.

# 1. Introduction

In the context of rapid world population growth and the steep decline in marine fisheries catches, the development of aquaculture is of significant interest for ensuring global food security and the conservation of wild marine stocks. In Europe, seabass (*Dicentrarchus labrax*) is currently one of the most farmed fish species and this high-value species is heavily traded (Bostock et al., 2016, FAO, 2018). Bacterial diseases such as pasteurellosis (*Photobacterium damselae* subsp. *piscicida*), photobacteriosis (*Photobacterium damselae* subsp. *damselae*), tenacibaculosis (*Tenacibaculum* spp.), and vibriosis (*Vibrio* spp.) represent severe threats to the seabass aquaculture sector (Buchmann et al., 2009, Muniesa et al., 2020). They may not only result in mass mortality events and highly negative economic effects, but are also potential zoonotic sources and may pose a risk for human health (Austin, 2010).

Among them, vibriosis, caused by certain species of *Vibrio*, stand as the most significant disease, affecting seabass at all stages of production chain (Le Breton, 1999, Pujalte et al., 2003, Korun et Timur, 2008, Muniesa et al., 2020). This disease is characterized by anorexia, erratic swimming behavior, exophthalmos, darkening of body color, pale gills, hemorrhages, ulcers, and skin lesions (Korun et Timur, 2008). Several *Vibrio* species have been reported in seabass infections: *Vibrio alginolyticus*, *Vibrio anguillarum*, *Vibrio harveyi*, *Vibrio ordalii*, *Vibrio parahaemolyticus* and *Vibrio vulnificus* (Ghittino et al., 2003, Kahla-Nakbi et al., 2006, Korun et Timur, 2008, Khouadja et al., 2013). *Vibrio anguillarum* has been the main significant seabass pathogens for
decades (Vázquez et Muñoz-Cueto, 2014). Commercially available prophylactic strategies, such as vaccines, have thus been extensively developed and effectively applied (Dadar et al., 2017). However, *V. harveyi* has recently been identified as an emerging major concern for seabass aquaculture and, to date, there are no specific, effective prophylactic control measures (Vendramin et al., 2016; Dadar et al., 2017).

*Vibrio harveyi* is a Gram-negative bacterium, ubiquitous in the marine environment, particularly in temperate and tropical waters. This species can be found in a free-swimming state, in biofilms attached to biotic and abiotic surfaces, or in symbiotic and host-pathogen interactions (Montánchez et Kaberdin, 2019). Importantly, *V. harveyi* can be pathogen for a wide range of cultivated hosts, including crustaceans, mollusks or fishes (Austin et Zhang, 2006). Its occurrence is likely to increase due to global warming and may lead to further *V. harveyi*-associated infections and have devastating effects for the aquaculture sector (Vezzulli et al., 2015). Interestingly, vibriosis outbreaks in seabass farms exhibit strong seasonality and are more likely to occur from June to November, when the water temperature is maximal (Pujalte et al., 2003). Warmer temperatures may indeed increase the abundance of viable *V. harveyi* in the surrounding environment and thus increase the prevalence of *V. harveyi* in fish (Kim et Lee, 2017). Importantly, contamination of fish usually occurs through contact with contaminated water via the digestive tract or skin (O'Toole et al., 2004). Although the development of a disease arises from the contamination of fish, the abundance of the pathogen in the surrounding environment may be an indicator of the risk of vibriosis. Other environmental factors such as salinity, fish density, pH, or the susceptibility and physiological state of the host, may have an impact on the development of vibriosis (Snieszko, 1974; Prayitno et Latchford, 1995; Vazzana et al., 2002; Maulvault et al., 2018). Moreover, biofilm formation on the walls of rearing tanks may enhance the risk of vibriosis outbreaks. Biofilms can facilitate resistance to antibiotics, and water sanitizers and may induce the persistence of potentially pathogenic bacteria in the aquaculture facility (Karunasagar et al., 1996; Bourne et al., 2006). Thus, the release of *V. harveyi* from biofilms in the rearing water or the contact of fish with the walls of aquaculture tanks may increase the prevalence of bacteria in fish and increase the risk of vibriosis outbreaks.
Therefore, monitoring the abundance of *V. harveyi* in the surrounding environment, such as in the rearing water and biofilms, may be an indicator of the *V. harveyi* contamination of seabass and thus of the probability of vibriosis outbreaks. Moreover, greater understanding of the correlation between the abundance, dynamics, and spatial patterns of *V. harveyi* and environmental biotic and abiotic factors might help to control vibriosis in aquaculture facilities.

Here, we report the dynamics and spatial patterns of cultivable Vibrionaceae and *V. harveyi*, monitored in 2018 from April to October (months when vibriosis is most likely to strike). The study was conducted on a semi-closed seabass farm, located in Northern France. The bacterial abundance and relative diversity were determined from water and biofilm in rearing tanks, and in the cold- and hot-water inlets supplying the farm. The dynamics of cultivable Vibrionaceae was assessed by culturing them on selective media at 37 °C. Presumptive cultivable Vibrionaceae were then identified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) using an in-house database, called Luvibase, previously and specifically designed to identify *Vibrio* species from the Harveyi clade (Mougin *et al.*, 2020b). The abundance of *V. harveyi* was estimated using a mreb-targeted real-time PCR (Mougin *et al.*, 2020a). Environmental parameters and fish density and/or mortality were recorded through the sampling campaign to identify the environmental factors that best correlate with bacterial abundance. Overall, the objective was to gain a greater understanding regarding the dynamics over time and spatial pattern of potentially pathogenic bacteria for seabass. This understanding can help design prophylactic measures to better manage vibriosis outbreaks.

## 2. Material and methods

### 2.1. Sampling site

This study was conducted from April to October 2018 in Aquanord Ichtus, a fish farm located in Gravelines, France along the southwestern coast of the North Sea. Because the aquaculture system is situated near a nuclear power plant, the site is monitored annually in order to detect any abnormalities in the development of the natural marine environment (Antajan *et al.*, 2019).
The farm is an intensive semi-closed system managed by a private company. This company currently raises seabass (*Dicentrarchus labrax*) and seabream (*Sparus aurata*) during the on-growing phase in flow-through tanks constructed from concrete. The water supply is regulated *via* two water inlets: a “cold-water inlet” from the natural marine environment and a “hot-water inlet” from the cooling system of a nuclear power plant nearby (Fig. 1). Water flow into the rearing tanks is regulated using a valve to maintain uniform water quality and temperature through the year. Overall, from mid-July to mid-September, the rearing tanks are supplied by the cold-water inlet only. During the rest of the year, the rearing tanks are supplied by both cold- and hot-water inlets. Fish are fed daily and aeration is provided to maintain an optimal level of dissolved oxygen (DO). Farmers use a commercial vaccine against *V. anguillarum* and an annual autogenous vaccine against *V. harveyi*. The latter is developed annually from pathogens isolated from the spleens of moribund fish during previous outbreaks.

Three similar rearing tanks were chosen for this study. The water capacity of each tank was 75 m³, with a water column of 1.3 m and a holding capacity of around 30 000 seabass. Average fish stock density was 48.13 kg.m⁻³, with an average fish weight of 200 g. During the sampling campaign, the aquaculture facility provided weekly biomass and mortality data from each rearing tank.

<figure><img src="image_44.png" /><figcaption>Figure 1. Schematic representation of the sampling areas on the fish farm, illustrating the sampling compartments and types of samples collected. The water samples were collected in the rearing tanks, and in the cold- and hot-water inlets every week. The biofilm samples were collected in the rearing tanks every two weeks.</figcaption></figure>
## 2.2. Sample collection and environmental parameters

Two types of samples, water and biofilm, were collected from three tanks containing seabass and from the cold- and hot-water inlets. Water samples were collected weekly from tanks and hot- and cold-water inlets. Biofilm samples were collected every two weeks from tanks. Both types of samples were harvested as previously detailed in Mougin et al. (2020a). Briefly, a volume of 1 L of water was collected in a sterile bottle in triplicate, from both water inlets and from the three rearing tanks, just below the surface of the water. Pieces of concrete, made of the same material used to build the tanks and placed 20 cm below the surface of the water, were collected in triplicate from the three rearing tanks (Supplementary Fig. 1). Samples were maintained on ice during transport to the laboratory. A total of 465 water samples and 144 biofilm samples were thus collected during the sampling campaign.

During sample collection, water temperature, pH, salinity, and conductivity were measured in each tank and in both inlets, within 50 cm of the water surface using a multi-parameter probe (Odeon Open X, Aqualabo, Champigny-sur-Marne, France). DO concentration was also measured in rearing tanks only using a Handy Polaris DO meter (OxyGuard, Farum, Denmark).

## 2.3. Sample preparation

Samples were processed as previously described in Mougin et al. (2019) and Mougin et al. (2020a). Briefly, water samples were filtered through 0.45 µm pore-size nitrocellulose filters (Sartorius, Göttingen, Germany). Filters were then immersed in 25 mL of Luria-Bertani broth supplemented with 20% NaCl (LBS) and 20% glycerol, and stored at -80 °C until analysis. Biofilms from the concrete samples were collected using a homemade sterile stainless steel jig by swabbing with two 2.34 cm² swabs. The swabs (150C, Copan, Brescia, Italy) were then immersed in 3 mL of LBS with 20% glycerol and stored at -80 °C until analysis. Subsequently, both types of samples were defrosted at room temperature and vortexed for 3 min to dislodge bacteria from the filters and swabs. Filters and swabs were removed the LBS-glycerol solution and the liquid was
centrifuged at 8000×g for 10 min. The cell pellets were then dissolved in 3 mL of physiological water (0.9% NaCl) and centrifuged at 8000×g for 10 min. Supernatants were removed and the cell pellets were dissolved in 2 mL of physiological water. Samples were then split into two subsamples. One subsample of 1800 µL was used to quantify *V. harveyi* using real-time PCR. The other 200 µL subsample was used for relative quantification and identification of presumptive cultivable Vibrionaceae by plating on selective agar media and by MALDI TOF MS.

## 2.4. Quantification of *V. harveyi* using real-time PCR

The 1800 µl subsample was centrifuged at 8000×g for 10 min and the supernatant was removed. Total DNA was extracted from the cell pellet, using the DNeasy PowerBiofilm kit (Qiagen, Hilden, Germany) in accordance with manufacturer's protocol, selected according to Mougin et al. (2020a). Relative quantification of *V. harveyi* based on a standard curve was performed according to the previously optimized real-time PCR protocol in Mougin et al. (2020a), using *V. harveyi*-specific primers targeting the *mreB* gene (synthesized by TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany). Briefly, pure DNA of the collection strain *V. harveyi* LMG 4044 was extracted using the DNeasy Blood&Tissue kit (Qiagen, Hilden, Germany). Purity was verified on a DeNovix DS-11 spectrophotometer (UV spectroscopy, Clinisciences, Nanterre, France) and the quantity was determined using a Qubit 3.0 fluorometer (dsDNA High-Sensitivity assay), (Invitrogen, Carlsbad, CA, USA). Then, standard curves were generated from a series of 10-fold dilutions of pure DNA of the collection strain *V. harveyi* LMG 4044. For each assay, the calibration curve was plotted using the means of the threshold cycle (C<sub>T</sub>) values from two replicate series versus the logarithm of each dilution factor. Each extracted environmental DNA template was analyzed at a 10-fold dilution to avoid the effects of real-time PCR inhibitors. Genome copy equivalent (GE) concentrations were then estimated, taking the dilution factor into account. All quantitative PCR reactions were run on a LightCycler® 480 (Roche Diagnostics, France) with a positive control (LMG 4044 *V. harveyi* DNA), a negative control (CIP 70.67 *V. campbellii* DNA), and a no template control (NTC), DNA-free. Melting curve analysis was performed to confirm the specificity of the fluorescent signals. Each extracted environmental DNA template was analyzed at least twice to ensure robust measurements.
## 2.5. Quantification of presumptive Vibrionaceae and identification using MALDI-TOF MS

An aliquot of 100 µL from the second subsample was plated on thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Biokar Diagnostics, Beauvais, France). The remaining 100 µL was serially 10-fold diluted in physiological water (10⁻¹, 10⁻², 10⁻³) and 100 µL of each dilution was plated onto TCBS agar. All plates were then incubated at 37 °C overnight. Presumptive cultivable Vibrionaceae colony forming units (CFUs) were counted using a Scan 500 plate reader (Interscience, Saint-Nom-la-Bretèche, France) and the concentration in each sample was estimated taking the dilution factor into account. Different colony morphotypes were screened on the basis of color (green or yellow), color shade, size, circumference (smooth or irregular) and presence/absence of a halo within 10 min after removal from the incubator. One colony of each different morphotype was randomly selected and incubated on LBS agar at 37 °C overnight. The morphological features of each selected colony were recorded. Pure bacteria were stocked in LBS broth supplemented with 20% glycerol at -80 °C until identification using MALDI-TOF MS.

Subsequently, once defrosted, these bacterial isolates were subcultured on marine agar (MA), (Difco Laboratories, Detroit, MI, USA) and incubated for 24 h, at 37 °C. Then, a single colony was plated on MA (Difco Laboratories) and incubated 24 h, at 37 °C to ensure the purity of the isolate. Preparation of bacterial extracts, target-loading and acquisition of MALDI-TOF MS profiles were performed as previously described in Mougin *et al.* (2020b). Briefly, the cellular proteins of each bacterial isolate were extracted from bacterial cells according to manufacturer's recommendations (Bruker France, Wissembourg, France). The extracts were stored at -20 °C until use. Then, 1 µL of the supernatant from each protein extract was deposited onto a clean 384-target ground steel microtiter plate (Bruker France) in four replicates. After air drying, 1 µL of a 10 mg.mL⁻¹ matrix solution of α-cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile and 2.5% trifluoroacetic acid (Sigma-Aldrich, St. Louis, MO, USA) was overlain on each spot. MALDI-TOF MS analysis was performed using an Autoflex SpeedTM (Bruker France), running Flexcontrol ver. 3.4 software (Bruker France). The Bruker bacterial test standard (BTS, Bruker
France) was used to calibrate the mass spectrometer before each run, according to the manufacturer's recommendations. Mass spectra were acquired in the positive linear ion mode across a mass/charge (m/z) ratio of 2000-20,000 Da. Mass spectra were acquired using the manufacturer's automatic MBT_FC.par method. For each MALDI-TOF MS profile, mass spectra were obtained from 5000 laser shots in 1000 shot steps performed randomly from different areas of the spot. Each MALDI-TOF MS profile was first compared with the mass spectra profiles (MSPs) of the Bruker BioTyper database ver. 9.0.0.0 implemented with the Luvibase, a supplemental, in-house *Vibrio* database (Mougin *et al.*, 2020b). The degree of concordance between experimental MALDI-TOF MS profiles and the MSPs was evaluated using a log-score calculated using the MALDI BioTyper Compass Explorer ver. 4.1 (Bruker France). A log-score exceeding 2.3 indicated a highly probable identification at the species level; a log-score between 2.0 and 2.3 indicated a probable identification at the species level; a log-score between 1.7 and 2.0 indicated a probable identification at the genus level only; and a log-score below 1.7 indicated no significant similarities between MSPs included in the databases and the unknown profile.

## 2.6. Statistical analysis

All statistical analyses were performed using RStudio ver. 3.5.2 software (R Core Team, 2013). Variation in bacterial abundance and environmental parameters were analyzed for each tank and inlet separately. A Kruskal-Wallis test for multiple comparisons and a Wilcoxon test for pairwise comparisons were used to analyze bacterial abundance between the three tanks and the hot- and cold-water inlets. A generalized linear mixed model with a random effect on time was used to reveal correlations between selected environmental factors: temperature, pH, salinity, conductivity, DO, fish data (i.e. biomass and mortality) and bacterial abundances. The models were constructed using the function lme in the nlme package ver. 3.1-137 (Pinheiro *et al.*, 2018) by excluding non-significant parameters or interactions one by one, starting with the interaction terms. For all tests, a p-value < 0.05 was considered statistically significant. All graphics were drawn using the ggplot2 packages ver. 2.2.1 (Wickham, 2016).
# 3. Results

## 3.1. Water quality and fish density and mortality

The values of the parameters describing water quality and fish density and mortality recorded from April to October 2018 are given in Table 1. In the cold-water inlet, the average temperature was 16.4 ± 3.5 °C and values fluctuated widely with a minimum of 9.1 °C, in April, and a maximum of 21.1 °C, in August. The pH, salinity, and conductivity did not vary widely and were 7.9 ± 0.3, 34.2 ± 0.4 and 51.9 ± 0.6 ms.cm⁻¹, respectively. In the hot-water inlet, average temperature was 23.8 ± 1.3 °C and values did not fluctuate widely, with a minimum of 19.9 °C in April and a maximum of 25.7 °C in September. The pH, salinity, and conductivity did not fluctuate widely either and were 7.9 ± 0.3, 34.6 ± 0.5 and 52.5 ± 0.7 ms.cm⁻¹ respectively. Temperature, pH, salinity and conductivity were similar among rearing tanks; the values were thus averaged. Average temperature was 19.1 ± 1.0 °C, with a minimum of 17.9 °C in April and a maximum of 21.1 °C in August. The pH, salinity, and conductivity did not vary widely and were 7.5 ± 0.3, 34.4 ± 0.5 and 52.3 ± 0.5 ms.cm⁻¹ respectively. During the sampling campaign, batches of fish were removed and replaced once from Tank 1 and once from Tank 2, due to their growing size. The average DO concentration in the rearing tanks was maintained at 6.05 ± 1.2 mg.L⁻¹. The mortality rate was low throughout the sampling campaign and no vibriosis outbreak was recorded in the three chosen rearing tanks.
<table><caption>Table 1. Means of parameters recorded from April to October 2018 in the aquaculture facilities.</caption><thead><tr><th>Sampling months</th><th>Temperature (°C)</th><th>pH</th><th>Salinity</th><th>Conductivity (ms.cm<sup>-1</sup>)</th><th>Dissolved oxygen (mg.L<sup>-1</sup>)</th><th>Fish density (kg.m<sup>-3</sup>)</th><th>Fish mortality per week (%)</th></tr></thead><tbody><tr><td>April</td><td>10.2</td><td>21.4</td><td>18.1</td><td>8.1</td><td>8.0</td><td>7.8</td><td>33.8</td><td>33.8</td><td>34.1</td><td>51.5</td><td>51.6</td><td>51.9</td><td>7.2</td><td>5.8</td><td>7.1</td><td>28.3</td><td>54.5</td><td>31.8</td><td>0.02</td><td>0.13</td><td>0.04</td></tr><tr><td>May</td><td>13.2</td><td>24.0</td><td>18.8</td><td>8.1</td><td>8.0</td><td>7.8</td><td>34.2</td><td>34.6</td><td>34.4</td><td>51.9</td><td>52.6</td><td>52.2</td><td>7.1</td><td>7.0</td><td>5.8</td><td>37.2</td><td>28.5*</td><td>34.2</td><td>0.01</td><td>0.02</td><td>0.01</td></tr><tr><td>June</td><td>17.0</td><td>24.3</td><td>19.2</td><td>7.8</td><td>7.9</td><td>7.5</td><td>34.1</td><td>34.6</td><td>34.2</td><td>51.9</td><td>52.5</td><td>52.0</td><td>5.9</td><td>5.4</td><td>5.4</td><td>42.8</td><td>39.5</td><td>37.5</td><td>0.00</td><td>0.08</td><td>0.29</td></tr><tr><td>July</td><td>20.0</td><td>24.1</td><td>20.2</td><td>7.8</td><td>7.8</td><td>7.4</td><td>33.8</td><td>34.1</td><td>33.9</td><td>51.5</td><td>51.9</td><td>51.6</td><td>5.6</td><td>5.5</td><td>5.4</td><td>52.5</td><td>47.0</td><td>35.5</td><td>0.01</td><td>0.01</td><td>0.04</td></tr><tr><td>August</td><td>20.3</td><td>24.0</td><td>20.4</td><td>8.0</td><td>7.9</td><td>7.5</td><td>34.5</td><td>34.6</td><td>34.8</td><td>52.3</td><td>52.5</td><td>52.4</td><td>6.1</td><td>6.4</td><td>5.5</td><td>54.0</td><td>55.8</td><td>43.6</td><td>0.03</td><td>0.04</td><td>0.06</td></tr><tr><td>September</td><td>18.0</td><td>24.1</td><td>18.4</td><td>8.1</td><td>8.0</td><td>7.4</td><td>34.4</td><td>35.4</td><td>34.9</td><td>52.2</td><td>53.6</td><td>53.0</td><td>6.4</td><td>5.4</td><td>5.6</td><td>62.8</td><td>67.0</td><td>53.0</td><td>0.02</td><td>0.01</td><td>0.04</td></tr><tr><td>October</td><td>15.8</td><td>24.7</td><td>18.5</td><td>7.7</td><td>7.7</td><td>7.1</td><td>34.6</td><td>34.8</td><td>34.6</td><td>52.5</td><td>52.8</td><td>52.6</td><td>7.5</td><td>5.0</td><td>5.7</td><td>51.3*</td><td>74.8</td><td>59.0</td><td>0.06</td><td>0.01</td><td>0.04</td></tr></tbody></table>

<footnote>*The on-growing batch of fish was removed and transported to a new rearing tank more suitable for their size, and a new batch of fish of smaller size was put in the rearing tank used in this study</footnote>
## 3.2. Identification of presumptive Vibrionaceae isolates cultured on TCBS agar

Across all the compartments, from April to October 2018, 1009 isolates were chosen on the basis of morphological traits on TCBS agar, after incubation at 37 °C for 24 h. Overall, 80.4% (811/1009) of the isolates were collected from the three rearing tanks: 44.2% (446/1009) originated from rearing water and 36.2% (365/1009) originated from biofilms. Only 11.5% (116/1009) and 8.1% (82/1009) were collected from the hot- and cold-water inlets, respectively (Fig. 2.a).

These isolates were then identified using MALDI-TOF MS with the Bruker BioTyper ver. 9.0.0.0 database supplemented with our in-house *Vibrio* Luvibase. This analysis resulted in the assignment of 91.2% (920/1009) of the isolates at the species level (log-score ≥ 2.0) and 8.8% (89/1009) at the genus level only (1.7 ≤ log-score < 2.0) (Fig. 2.b). Among the 920 isolates identified at the species level, 75.7% belonged to genus *Vibrio* and 24.3% to *Photobacterium*. Within the *Photobacterium* genus, only *P. damselae* was isolated. Within the *Vibrio* genus, the isolates were categorized into four clades, the Harveyi clade (97.3%), the Splendidus clade (2.0%), the Orientalis clade (0.6%) and the Coralliilyticus clade (0.1%). The Harveyi clade consisted of isolates assigned to *V. alginolyticus*, *V. harveyi*, *V. owensii*, *V. rotiferianus*, *V. jasicida*, *V. mytili*, *V. campbellii* and *V. parahaemolyticus*. The Splendidus clade consisted of isolates identified as *V. pelagius* and *V. fortis*. The Orientalis clade consisted of isolates assigned to *V. brasiliensis*, and *V. sinaloensis*. The Coralliilyticus clade consisted of one isolate assigned to *V. coralliilyticus*. Overall, the most frequently identified species were *V. alginolyticus* (24.8%), *P. damselae* (24.3%), *V. harveyi* (24.2%) and *V. owensii* (15.8%) (Fig. 2.b). All other species were very scarce.
a.

<figure><img src="image_45.png" /></figure>

b.

<figure><img src="image_46.png" /></figure>

c.

<figure><img src="image_47.png" /></figure>

d.

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>

<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /></figure>

<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /></figure>

Figure 2. Qualitative analysis of the distribution and identification of the 1009 isolates (TCBS agar, 37 °C), using MALDI-TOF MS. a. Relative proportions of the isolates collected from the different sampling sites: rearing tanks and hot-/cold- water inlets and different sample types: water and biofilm. b. Relative proportions of identified species regardless of the sampling site and sample type. c. Relative proportions of identified species in the different sampling sites and sample types. d. Identified species in the different sampling sites and sample types throughout the sampling campaign, from April to October 2018. The key on the right indicates the color codes of the identified species, ranked from the most abundant (top) to the least abundant (bottom).
The relative isolated and identified bacterial diversity was roughly similar between the compartments and sample types (Fig. 2.c). Nevertheless, none of the species were isolated systematically throughout the sampling campaign (Fig. 2.d). *Vibrio alginolyticus* was recorded from May to October in the hot-water inlet, water tanks and biofilm, but was not recorded in June and October in the cold-water inlet. The presumed pathogen *V. harveyi* was recorded from May to September in all compartments. *Photobacterium damselae* was recorded from July to October in the hot- and cold-water inlets and from May to October in the water tanks and biofilms. Finally, *V. owensii* was recorded from May to October in water tanks and biofilms, but recorded from July to September in the hot-water inlet, and in July and August only in the cold-water inlet.

### 3.3. Quantification of presumptive culturable Vibrionaceae isolates

Presumptive culturable Vibrionaceae CFUs grown on TCBS agar, after incubation at 37 °C for 24 h, were enumerated and their abundance in each compartment and each sample was estimated. According to the statistical analysis, the average abundance of presumptive culturable Vibrionaceae differed significantly between the cold- and hot-water inlets and water from rearing tanks (each p-value < 0.05). In contrast, there were no significant differences between the average abundances of the three rearing tanks, for either the water or biofilm samples (each p-value > 0.05). Consequently, for the following analyses, for each sampling date, the average abundances detected in the three rearing tanks were pooled.

In the cold-water inlet, the presumptive detected culturable Vibrionaceae, ranged from 3.37×10² to 4.85×10³ CFU.mL⁻¹ (Fig. 3.a). The maximum abundance was detected from July to September, when the temperature recorded in this compartment was the highest, 19.8 ± 1 °C. A peak in abundance (3.37×10² CFU.mL⁻¹) was observed in late May at a temperature of 15.0 °C, and water temperature continued to increase slightly (average + 0.7 °C each week from April to June). No bacteria were detected in October and April.
In the hot-water inlet, the presumptive detected culturable Vibrionaceae followed more or less the same pattern, ranging from 3.11×10² to 6.71×10⁴ CFU.mL⁻¹ in abundance. Maximum abundance was detected in late June when the temperature reached 24 °C. Then, abundance tended to decrease slightly until mid-October, when a peak of 5.99×10² CFU.mL⁻¹ was detected at a temperature of 24.9 °C. As in the cold-water inlet, a peak in abundance (6.43×10³ CFU.mL⁻¹) was detected in late May at a water temperature of 24.7 °C in this compartment.

In the rearing tanks, the presumptive culturable Vibrionaceae detected in water ranged from 2.28×10³ to 1.74×10⁵ CFU.mL⁻¹. Presumptive detected culturable Vibrionaceae were enumerated from mid-April until the end of the study. The abundance tended to increase slowly and constantly with an average + 0.20 log per week until August, the warmest month of summer with the maximum recorded temperature (21.7 °C). Subsequently, abundance decreased slightly in September, and hit a peak of 2.50×10⁴ CFU.mL⁻¹ in mid-October, when the temperature jumped back to 19.1 °C. The presumptive attached culturable Vibrionaceae detected in biofilms ranged from 2.51×10⁵ to 2.03×10⁷ GE.cm⁻². Presumptive attached culturable Vibrionaceae were enumerated from mid-May until the end of the study. As observed in the water samples, abundance tended to increase slowly and constantly with an average + 0.27 log per week until August, then decreased slightly. Interestingly, a peak in the abundance of 2.29×10⁶ GE.cm⁻² occurred with a peak in temperature in mid-October, similar to the pattern for the water samples and the hot-water inlet.
a. Presumptive Vibrionaceae - TCBS agar counts

<figure><img src="image_56.png" /></figure>

b. Vibrio harveyi - real-time PCR quantification

<figure><img src="image_57.png" /></figure>

Figure 3. Fluctuation in abundance of a. presumptive culturable Vibrionaceae on TCBS agar, 37 °C, and b. Vibrio harveyi in the cold- and hot-water inlets, water and biofilm samples from rearing tanks. For inlet samples, abundance values are the mean of three samples, collected simultaneously. For rearing tanks, abundance values are the mean of nine replicates, corresponding to the three replicates collected simultaneously from each of the three rearing tanks. The area around the curves represent the 95% confidence intervals for the abundance index. Water temperature in all compartments are also shown.
3.4. Quantification of *V. harveyi* using real-time PCR

Real-time PCR was used to estimate the abundance of *V. harveyi* detected in each compartment and each sample. According to the statistical analysis, the average abundance of presumptive culturable Vibrionaceae detected in the water from the cold- and hot-water inlets and rearing tanks were significantly different (each p-value < 0.05). In contrast, there were no significant differences between the average abundances observed in the three rearing tanks, for either the water or biofilm samples (each p-value > 0.05). Consequently, for each sampling time, the average abundances detected in the three rearing tanks were pooled together.

Overall, the average estimated quantity of *V. harveyi* was higher in hot-water inlet than in the cold-water inlet. In the cold-water inlet, abundance of detected *V. harveyi*, ranged from 1.35 to 8.57 GE.mL⁻¹ (Fig. 3.b). A few peaks were detected in late May, mid-June, early July, and mid-September. Successive screenings for *V. harveyi* over 5 weeks demonstrated a rapid increase in abundance from mid-July to mid-August, when temperatures reached their highest values.

In the hot-water inlet, abundance of detected *V. harveyi* ranged from 4.62 to 2.11×10² GE.mL⁻¹.

*V. harveyi* was first detected in mid-May when the temperature reached 25.1 °C for the first time in the year. Abundance increased slowly (average + 0.11 log per week) until early June when the concentration reached 1.41×10² GE.mL⁻¹, and then decreased. The average abundance detected in late June and mid-July was relatively high, with abundance decreasing slightly thereafter (average + 0.20 log per week), dropping to an absence of detection until a last peak of 4.62 GE.mL⁻¹ in mid-October when the temperature reached 24.9 °C.

In the rearing tanks, detected *V. harveyi* abundance ranged from 3.72 to 9.51×10¹ GE.mL⁻¹ (Fig. 3.a). *V. harveyi* was first detected in April when the temperature reached 18.0 °C. The maximal abundances were recorded in mid-May (9.51×10¹ GE.mL⁻¹), mid-July (7.88×10¹ GE.mL⁻¹), and mid-October (7.65×10¹ GE.mL⁻¹), when the rearing water temperatures reached 19.0, 19.6 and 19.1 °C, respectively. Nevertheless, *V. harveyi* was detected in April when no *Vibrio* bacteria were recorded in the cold- and hot-water inlets. The abundance of attached *V. harveyi* detected
in biofilms ranged from 1.45×10³ to 3.25 ×10³ GE.cm⁻² (Fig. 3.b). Attached *V. harveyi* were enumerated from mid-May (1.64 ×10³ GE.cm⁻²) until mid-October (2.43×10³ GE.cm⁻²), with no detection in early September or in early October.

**3.5. Influence of environmental factors on cultivable Vibrionaceae and *V. harveyi* dynamics**

Statistical analysis highlighted correlations between water temperature and the abundance of presumptive Vibrionaceae in the cold-water inlet (p-value = 0.0065). A positive correlation was detected between water temperature (p-value = 0.0005) and the abundance of presumptive Vibrionaceae in biofilms.

Moreover, statistical analyses highlighted a positive correlation between water temperature and the abundance of *V. harveyi* in the cold-water inlet (p-value=0.0186) and in water from rearing tanks (p-value = 0.0198). A positive correlation was also detected between water temperature and the abundance of *V. harveyi* in biofilms (p-value < 0.0001).

**4. Discussion**

The seabass aquaculture sector is being challenged by the emergence of recurring vibriosis outbreaks caused by *V. harveyi* (Vendramin et al., 2016). The prevalence of the pathogen in fish is highly correlated with the abundance of the pathogen in the surrounding environment (Kim et Lee, 2017). Although the contamination of fish is not the only factor that can exacerbate the development of the disease, the abundance of *V. harveyi*, in rearing water and biofilm may be an accurate indicator of the risk of vibriosis outbreaks. Thus, deeper understanding of the dynamics and spatial patterns of *V. harveyi* in the aquaculture environment is needed.

In this study, we assessed the temporal and spatial dynamics of Vibrionaceae, and the presumptive pathogen *V. harveyi* in particular, from April to October 2018 on a fish farm located in Northern France. Three rearing tanks were chosen from a facility frequently affected by vibriosis. In particular, vibriosis outbreaks, mostly caused by *V. harveyi*, were recorded in 2017
on this fish farm. Furthermore, a few strains of *V. jasicida*, *V. rotiferianus*, *V. owensii*, *V. tasmaniensis*, and *V. gigantis* were also isolated from the spleens of moribund seabass (Mougin *et al.*, 2020b, Mougin *et al.*, 2020a). In 2018, vibriosis outbreaks caused by *V. anguillarum* were recorded (Supplmentary Table 1). According to the aquaculture facility operators, these latter outbreaks were due to a delay in vaccine delivery and were quickly and successfully managed. Some strains of *V. harveyi* were isolated from few moribund fish (Supplmentary Table 1), but no *V. harveyi* vibriosis outbreak has been recorded on the fish farm, particularly in the three rearing tanks used during our monitoring study.

Among potentially pathogenic bacteria for seabass, *P. damselae*, *V. alginolyticus*, *V. owensii*, *V. rotiferianus*, and *V. harveyi* were isolated during our sampling campaign. The species *P. damselae* is divided into two subspecies with clinical and epidemiological differences, both affecting seabass (Vázquez et Muñoz-Cueto, 2014, Essam *et al.*, 2016). *P. damselae* subsp. *piscicida* (formerly *Pasteurella piscicida*) is known as the causative agent of a severe aquatic disease called pasteurellosis, and *P. damselae* subsp. *damselae* (formerly *Vibrio damsela*) is responsible for photobacteriosis commonly defined as sporadic pathologies (Buchmann *et al.*, 2009). Nonetheless, this latter disease had been recognized as an epizoonotic agent, transmitted by infected fish, and may cause public health problems (Rivas *et al.*, 2013). In our study, presumably, only subspecies *damselae* was recorded, but the MALDI BioTyper database cannot discriminate between these two subspecies (Pérez-Sancho *et al.*, 2016). Given that no mortalities caused by *P. damselae* have been recorded, no further analysis was pursued. In the *Vibrio* genus, the Harveyi clade, especially *V. alginolyticus*, *V. harveyi*, *V. owensii*, and *V. rotiferianus* were the most frequently isolated species on the fish farm. Importantly, these species have already been isolated from moribund seabass and are considered as potential pathogens on the farm. Nevertheless, *V. harveyi* remains the main pathogen responsible for recurrent vibriosis outbreaks.

Interestingly, even though no vibriosis outbreak was recorded, isolates of *V. harveyi* were detected in the rearing tanks from water and biofilm, throughout practically the whole sampling campaign. The development of vibriosis occurs due to disruptions between host-pathogen-
environment interactions (Snieszko, 1974, Toranzo et al., 2005). Thus, many factors, such as the susceptibility and physiological state of the host, the pathogenicity and virulence of the pathogen, and the influence of environmental stress conditions, must be considered. At this fish farm, the absence of vibriosis outbreaks was attributed to the efficiency of the novel annual autogenous vaccine. However, in practical terms, this type of vaccine sometimes appears less efficient from one year to the next and must be regularly renewed, incurring high costs. Another hypothesis is that the strains of *V. harveyi* present on the farm during 2018 were not highly virulent. Our study focused on the abundance of all *V. harveyi* strains without distinguishing pathogens from non-pathogens. There is thus a possibility that the pathogenic isolates detected and collected in 2018 are scarce.

Environmental factors may also have an impact on the development of the disease. The correlation between the abundance of *V. harveyi* and water temperature indicates that the risk of vibriosis outbreaks may vary throughout the year. In the natural marine environment, previous studies have highlighted that the abundance of *Vibrio* spp. may follow seasonal variation in temperate regions (Martinez-Urtaza et al., 2008, Johnson et al., 2012). Water temperature and salinity are considered as the most effective predictors of temporal and spatial distributions. However, in a semi-closed aquaculture system, even though tanks are supplied with natural marine seawater, water quality is extensively monitored and maintained as constant as possible. Consequently, in our study, the environmental parameters monitored in rearing tanks (i.e. pH, salinity, conductivity, and DO) did not vary widely. Water temperature was generally constant, but the month of June and July showed temperatures above 20 °C. Increased prevalence of *V. harveyi* in oysters (*Crassostrea gigas* and *Crassostrea virginica*) was detected at temperatures above 20 °C (Pujalte et al., 1999). Moreover, a previous study (Pujalte et al. (2003) reported increased prevalence of *V. harveyi* in European seabass and gilthead seabream during warm months. Consequently, the warmer months of summer may represent a higher risk for vibriosis outbreaks. Furthermore, temperature may not only influence the prevalence of the pathogen, but also the virulence and the physiological state of the host, disturbing the host-pathogen relationship (Paillard et al., 2004, Travers et al., 2009). In the years ahead, the risk of vibriosis