# Bacteria with antibacterial activities isolated from Magallana gigas microbiota as potential probiotics against Vibrio aestuarianus infections in oyster farming

Luc Dantan, Marie-Agnès Travers, Lionel Dégremont, Benjamin Morga, Prunelle Carcassonne, Mickael Mege, Yannick Fleury, Bruno Petton, Elise Maurouard, Jean-François Allienne, et al.

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## ► To cite this version:

Luc Dantan, Marie-Agnès Travers, Lionel Dégremont, Benjamin Morga, Prunelle Carcassonne, et al. Bacteria with antibacterial activities isolated from Magallana gigas microbiota as potential probiotics against Vibrio aestuarianus infections in oyster farming. Aquaculture, 2025, 609, pp.742949. 10.1016/j.aquaculture.2025.742949. hal-05172277

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# Bacteria with antibacterial activities isolated from *Magallana gigas* microbiota as potential probiotics against *Vibrio aestuarianus* infections in oyster farming

Luc Dantan<sup>a</sup>, Marie-Agnès Travers<sup>b</sup>, Lionel Degremont<sup>c</sup>, Benjamin Morga<sup>c</sup>, Prunelle Carcassonne<sup>a</sup>, Mickael Mege<sup>c</sup>, Yannick Fleury<sup>d</sup>, Bruno Petton<sup>e</sup>, Elise Maurouard<sup>c</sup>, Jean-François Allienne<sup>a</sup>, Gaëlle Courtay<sup>b</sup>, Océane Romatif<sup>b</sup>, Raphaël Lami<sup>f</sup>, Laurent Intertaglia<sup>g</sup>, Yannick Gueguen<sup>h</sup>, Jeremie Vidal-Dupiol<sup>b</sup>, Céline Cosseau<sup>a</sup>, Eve Toulza<sup>a,\*</sup>

## ARTICLE INFO

**Keywords:**

*Magallana gigas*

Antibacterial activities

Microbiota

*Vibrio aestuarianus*

Aquaculture

## ABSTRACT

**Introduction:** Oyster farming is a significant industry worldwide, but it is threatened by various diseases such as Pacific Oyster Mortality Syndrome and vibriosis. *V. aestuarianus* is one of the major causes of mortality for market-size oysters, resulting in significant economic losses for oyster farmers. Among the various control methods developed, probiotics appear to be a promising approach. More specifically, the use of the antibacterial activity of bacteria from the natural microbiota of the oyster *Magallana gigas* appears to be a sustainable solution against *V. aestuarianus* infections.

**Results:** Our study investigated the probiotic potential of bacteria isolated from the microbiota of *M. gigas* oysters. We screened a collection of 334 bacteria against eight target pathogens, including *V. aestuarianus*, and identified 78 bacteria with antibacterial activity for which eight retained this activity in their culture supernatants. Five strains were selected for further testing and exposed to oysters prior to *V. aestuarianus* infection. Our results show that four strains significantly reduced oyster mortality, with a maximum reduction of 70 %. In addition, changes in oyster microbiota composition were observed following exposure, but the administered bacteria were not detected in the microbiota.

**Conclusion:** Our findings demonstrate the potential of oyster microbiota-derived bacteria as probiotics for disease control in oyster farming. This approach could provide a sustainable and environmentally friendly solution for the oyster farming industry. Further research is needed to understand the underlying mechanisms and to develop effective probiotic-based strategies for preventing *V. aestuarianus* infection.

## 1. Introduction

The Pacific Oyster *Magallana gigas* (formerly known as *Crassostrea gigas*), is the most widely cultivated oyster species in the world,

contributing significantly to the aquaculture industry (Food and Agri-culture Organisation, 2022). Nevertheless, the farming of *M. gigas* encounters substantial difficulties due to recurrent infectious diseases, leading to high annual mortality rates (Azéma et al., 2015; Cotter et al.,

<footnote><sup>a</sup> IHPE, Univ Perpignan Via Domitia, CNRS, IFREMER, Univ Montpellier, Perpignan, France</footnote>

<footnote><sup>b</sup> IHPE, Univ Montpellier, CNRS, IFREMER, Univ Perpignan Via Domitia, Montpellier, France</footnote>

<footnote><sup>c</sup> Ifremer, ASIM, F-17390 La Tremblade, France</footnote>

<footnote><sup>d</sup> Laboratoire de Biotechnologie et Chimie Marine, EA3884, Université de Bretagne Occidentale, Université Bretagne Sud, 29334 Quimper, France</footnote>

<footnote><sup>e</sup> Univ Brest, Ifremer, CNRS, IRD, LEMAR, F-29280, Plouzané, France</footnote>

<footnote><sup>f</sup> Sorbonne Université, UPVD, CNRS, Laboratoire de Biodiversité et Biotechnologies Microbiennes, F-66650 Banyuls-sur-Mer, France</footnote>

<footnote><sup>g</sup> Sorbonne Université, CNRS, Bio2Mar, Observatoire Océanologique, 66650 Banyuls-sur-Mer, France</footnote>

<footnote><sup>h</sup> MARBEC, Univ Montpellier, CNRS, Ifremer, IRD, Sète, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: eve.toulza@univ-perp.fr (E. Toulza).</footnote>
2010; Friedman et al., 2005; Pernet et al., 2012). Since 2001, mass mortality of adult *M. gigas* has been reported in France, in association with the detection of the bacterium *Vibrio aestuarianus* (Garnier et al., 2008). This bacterium is a harmful primary pathogen with chronic mortality reaching a cumulative mortality rate up to 30 %. This represents important economic consequences since *V. aestuarianus* preferentially infects market size oysters which have been raised for several years (Azéma et al., 2017; Lupo et al., 2019). Other *Vibrio* species have been associated with mortality episodes affecting *M. gigas* oysters at different stages of development. Notably, *V. coralliilyticus* has been linked to massive mortalities of *M. gigas* larvae (Richards et al., 2015; Travers et al., 2015; Ushijima et al., 2022). Spat / juveniles are affected by *V. crassostreae* (Cowan et al., 2023; Dégremont et al., 2021) and *V. harveyi* (Dégremont et al., 2021; Oyanedel et al., 2023).

Efforts to address the challenges of *Vibrio* infections affecting *M. gigas*, have led to various strategies build upon growing knowledge on oysters. One promising method involves the use of genetic selection to breed pathogen-resistant oysters (Dégremont et al., 2015, 2020), although this approach has limitations, such as the potential selection of trade-offs that could negatively impact the commercial value of *M. gigas*. Furthermore, the discovery of immune priming in *M. gigas* has paved the way for innovative applications using heat-killed *V. splendidus* (Zhang et al., 2014), which provide protection against *V. splendidus* infections.

Research on disease prevention in molluscs based on the use of probiotics has been ongoing for decades (Dantan et al., 2024a, 2024b; Muñoz-Cerro et al., 2024; Takyi et al., 2023, 2024; Yeh et al., 2020). Probiotics display their positive benefits through a variety of methods, including direct pathogen inhibition via competition for nutriments or production of antimicrobial compounds, but also indirect immunomodulatory effects (Khademzade et al., 2020; Lazado and Caipang, 2014; Peixoto et al., 2017; Yan et al., 2014). Previous studies have demonstrated that bacterial strains *Pseudoalteromonas* sp. hCg-6 and *Pseudoalteromonas* sp. hCg-42 isolated from *M. gigas* haemolymph, displayed in vitro antibacterial activity against marine pathogens such as *V. splendidus*, *V. tapetis*, *V. harveyi* and *Aeromonas salmonicida* (Defer et al., 2013; Desriac et al., 2014; Offret et al., 2018). Furthermore, exposure to *Pseudoalteromonas* sp. hCg-6 has been shown to enhance the survival of *Haliotis tuberculata* abalone during infection with *V. harveyi* ORM4 (Offret et al., 2018). In addition, antimicrobial-producing bacteria have been employed as a strategy to improve the survival of oysters against bacterial infections. For instance, *Crassostrea virginica* larvae exposed to *Bacillus pumilus* RI06-95 exhibited significantly increased survival rates during challenge with *Vibrio coralliilyticus* (Sohn et al., 2016). Similarly, an exposure of *M. gigas* larvae to *Pseudoalteromonas* sp. was found to inhibit the growth of *Vibrio coralliilyticus*, thereby improving larval survival during subsequent infection (Madison et al., 2022). Probiotic strategies have thus shown promise in improving oyster resistance to bacterial infections through microbial competition and immune modulation.

Existing probiotic strategies commonly rely on allochthonous bacterial strains, selected for their beneficial traits such as antimicrobial activity, immune stimulation, or improved nutrient assimilation. While effective in some contexts, these strains may not be optimally adapted to aquaculture farming. Native microbiota isolates are in contrast inherently adapted to their host and environmental conditions, which can enhance their ecological compatibility and functional efficacy while reducing the risk of unintended ecological impacts.

In this article, we investigated the potential of bacteria isolated from the natural microbiota of *M. gigas* to protect oysters against *V. aestuarianus* infection, hypothesising that these microbiota-derived strains would confer protection through direct antagonism effects or indirect protective effect against the pathogen. For this, we firstly screened a collection of bacteria previously isolated from *M. gigas* associated microbiota (Dantan et al., 2024a) for their antibacterial activity in vitro against four oyster pathogenic *Vibrio* sp. (Travers et al., 2015) and against opportunistic bacteria associated with the Pacific

Oyster Mortality Syndrome (POMS) disease (Clerissi et al., 2022; de Lorgeril et al., 2018). Secondly, selected candidate bacteria were tested for their effect against *V. aestuarianus* infection, and we investigated the impact of the administered bacteria on the microbiota of the exposed oysters.

## 2. Materials and methods

### 2.1. Screening for antibacterial activities of bacteria isolated from *M. gigas* microbiota

Eight bacterial strains were selected as targets for the screening of antibacterial activities. Four of them were pathogenic *Vibrio* for oysters at different developmental stages: *Vibrio aestuarianus* 02/041 (Garnier et al., 2008), *Vibrio coralliilyticus* 06/210 (Dégremont et al., 2021), *Vibrio crassostreae* J2-9 (Lemire et al., 2015) and *Vibrio harveyi* Th15_O_A01 (Oyanedel et al., 2023). The four others are bacteria associated with POMS dysbiosis according to (Clerissi et al., 2022; de Lorgeril et al., 2018): *Amphitrea* sp. 14/114-3 T2, *Marinobacterium* sp. 05/091-3 T1, *Marinomonas* sp. 12/107-2 T2, *Pseudoalteromonas* sp. 09/041-1 T3 (Supplementary_File_1 Table S1). Target bacteria were provided by the French National Reference Laboratory (Ifremer, La Tremblade, France) or came from previous projects carried out in our laboratory (Oyanedel et al., 2023).

All strains were cultivated from glycerol stock in 10 mL Marine Broth (MB) at 20 °C for 48 h under constant agitation (100 rpm), then the OD600 was determined using BioPhotometer (Eppendorf). The cultures were diluted into fresh MB to a final concentration of 10⁶ CFU/mL prior to inoculation of Marine Agar plates by inundation. The 334 bacteria from our previously described bacterial collection isolated from *M. gigas* microbiota (Supplementary_File_1 Table S2) (Dantan et al., 2024a) were then tested for their antibacterial activity against each of the target. Each bacteria from the collection were grown from glycerol stock in 2 mL MB at 20 °C under constant agitation (100 rpm) for 48 h before being distributed onto 4 different 96 well microplates. These microplates were duplicate using a microplate pin replicator on new 96 well microplate containing fresh MB. After overnight incubation at 20 °C under constant agitation (100 rpm), 2 µL from each well was transferred (in 8 × 12 array) onto marine agar plate previously inoculated with the target bacteria. As control a 2 µL spot of kanamycin (50 µg/mL) served as a positive control, and a 2 µL spot of sterile Marine Broth served as a negative control (Laguerre et al., 2025). Marine agar plates without target bacteria were used to assess the growth and purity of the bacteria from collection. Agar plates were then incubated at 20 °C for two days and were then photographed using Gel Doc XR (Biorad, CA, USA) and the "Flamingo" filter to visualise a potential halo of inhibition characteristic of antibacterial activity. For supernatant assay, the same target bacteria were used. Prior to the test, the bacteria were cultured in Marine Broth media on 96 well plates during 72 h at 20 °C. After the incubation period, the 96 well plate was centrifugated for 10 min at 4000 rpm. The supernatants were then carefully transferred into new 96 well plates and heated at 100 °C for 5 min to kill the possible remaining bacteria. Then, 2 µL spots were deposited on the marine agar plates previously flooded with the target bacteria as describe above.

### 2.2. Oysters used as donors and recipient

Oysters were produced at the Ifremer hatchery in La Tremblade in February 2021. Briefly, 25 females and 25 males were used to produce 100 bi-parental families (each male was mated to four females, and each female was mated to four males).

Each family was raised in separated tank during the larval stage, and then each family was settled in separate trays until two-months old. Then, 150 spat of each family were individually counted and mixed together to produce a batch. This batch of mixed families was transferred to the Ifremer nursery in Bouin in May 2021 until the
experimental infection in November 2021. All oysters were kept in our controlled facilities using UV-treated seawater until their evaluations. Animals were fed ad libitum using a cultured phytoplankton diet (Isochrysis galbana, Tetraselmis suecica and Skeletonema costatum).

## 2.3. Oyster exposure to bacteria selected for a potential beneficial effect

Recipient oysters were distributed between seven 40 L tanks filled with UV-treated seawater and maintained at 20 °C with adequate aeration. Each tank contained 75 adult oysters (mean individual weight = 29.68 ± 8.03 g). These recipient oysters were either exposed to one of the five bacterial strains selected for their antibacterial activities, one control bacteriocin producing strain (*Pseudoalteromonas* sp. hCg-42) which has been previously isolated from *M. gigas* haemolymph (Defer et al., 2013) or to sterile artificial seawater (control).

Prior to exposure, the bacteria (*Pseudoalteromonas* sp. hCg-42, *Bacillus* sp. ARG61, *Halomonas* sp. LTB66, *Cytobacillus* sp. ARC29, *Yoonia* sp. THAU59 and *Vibrio* sp. LTB1) were individually cultured in 10 mL of MB media for 48 h at 20 °C under constant agitation. Then, 1 mL of each bacterial culture was inoculated into 10 mL fresh MB media and incubated at 20 °C under constant agitation. After 48 h of incubation, the OD₆₀₀ was measured, and the appropriate amount of bacteria (1 OD₆₀₀ unit = 8 × 10⁸ CFU/mL) was collected, centrifuged at 4000 rpm for 2 min and the supernatant was discarded. The pellets were then resuspended in 10 mL sterile artificial seawater and added immediately to tanks containing the adult oysters so that the final concentration in the tank was adjusted to 10⁴ CFU/mL. The selected bacteria were added individually 3 times on days zero, two and four (D0, D2 and D4), without water renewal, resulting in a 7-days balneation exposure period for the oysters(Fig. 1).

Five oysters exposed to each condition were sampled at day seven (D7) of the bacterial exposure to perform microbiota sequencing. Sampled oysters were grounded in liquid nitrogen (Retsch MM400 mill) to a powder that was stored at -80 °C until subsequent DNA extraction.

## 2.4. Vibrio aestuarianus experimental infection by cohabitation

A *V. aestuarianus* experimental infection was performed immediately following the bacterial exposure to recipient oysters. A cohabitation protocol was used as previously described in (De Decker and Saulnier, 2011). The *V. aestuarianus* 02/041 was grown in Zobell medium at 22 °C for 24 h under agitation. The bacterial concentration was determined by measuring the OD₆₀₀ and was adjusted to OD₆₀₀ of 1 representing 5 × 10⁸ bacteria per mL in artificial seawater. Seventy donor oysters were injected in the adductor muscle with 100 µL of the *V. aestuarianus* 02/041 suspension and were then equally distributed among the seven tanks (Fig. 1). The donors were from the same oyster population as the recipient oysters exposed to the strains showing antibacterial activities. A ratio 1:1 (70 donor oysters for 70 recipient oysters per tank) was used for donor and recipient oysters (Azéma et al., 2017). After 48 h of cohabitation and before first mortality, donors were removed from the tanks. The mortality of recipient oysters exposed to selected bacteria and control oysters, was then recorded during 17 days by conting and removing the dead oysters every day.

## 2.5. Statistical analysis of oyster mortality

Oyster mortality was Analysed using survival analysis performed on R (v 4.2.1) (R Core Team, 2022) with the package survminer (v 0.4.9) (https://cran.r-project.org/web/packages/survminer/index.html). The Kaplan-Meier method was used to represent the cumulative survival rate. A log-rank test was used to determine the difference between the conditions and post-hoc pairwise comparisons with Bonferroni corrected *p*-value were used to define which values were significantly different from the control. A multivariate Cox proportional hazards regression model was used to compute Hazard-Ratio (HR) with

<figure><img src="image_4.png" /><figcaption>**Fig. 1. Overall experimental design for bacterial exposure and experimental infections performed with oysters exposed to selected bacteria.**
Recipient oysters (n = 75) were placed in 40 L tank filed with UV-treated seawater and maintained at 20 °C with adequate aeration. Oysters were then exposed to individual selected bacterial strains during seven days with a renewal every two days (indicated by the “+” sign in the green circle). At the end of bacterial exposure, 5 oysters per tank were sampled, flash-frozen into liquid nitrogen and stored at -80 °C for molecular analysis (indicated by red triangles). Right after the bacterial exposure, remaining recipient oysters were transferred into 5 new tanks containing donor oysters injected with *V. aestuarianus* in order to realise an experimental infection. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

confidence intervals of 95 %.

## 2.6. Bacteria and oyster DNA extraction

DNA extraction from oysters collected during bacterial exposure was performed from frozen powders using DNA from the tissue Macherey-Nagel kit according to the manufacturer's protocol. Prior to 90 min of enzymatic lysis in the presence of proteinase K, an additional 12-min mechanical lysis (Retsch MM400 mill) was performed with zirconia/silica beads (BioSpec). DNA concentrations were checked with a Qubit® 2.0 Fluorometer (Thermo Scientific) and adjusted when necessary.

Bacterial DNA for the candidate strains used to constitute the mock community was extracted as described in (Dantan et al., 2024a).
## 2.7. 16S rDNA library construction and sequencing

Library construction (with primers 341F 5'-CCTAYGGGRBGCASCAG and 806R 5'-GGACTACNNGGGTATCTAAT targeting the 16S V3V4 region) and sequencing on a MiSeq v2 (2 × 250 bp) were performed by ADNid (France).

## 2.8. Bioinformatic pipeline for 16S barcoding analysis

Sequencing data obtained in this study were processed with the SAMBA (v 3.0.2) workflow developed by the SeBiMER (Ifremer's Bioinformatics Core Facility). Briefly, Amplicon Sequence Variants (ASV) are constructed with DADA2 (Callahan et al., 2016) and the QIIME2 dbOTU3 (v 2020.2) tools (Bolyen et al., 2019), Due to the known diversity overestimation generated by DADA2, an additional step of ASV clustering has been performed using dbOTU3 algorithm (Olesen et al., 2017) and contaminations were removed with microDecon (v 1.0.2) (McKnight et al., 2019). Taxonomic assignment of ASVs was performed using a Bayesian classifier trained with the Silva database v.138 using the QIIME feature classifier (Wang et al., 2007). Finally, community analysis and statistics were performed on R (R version 4.2.1) (R Core Team, 2022) using the packages phyloseq (v 1.40.0) (McMurdie and Holmes, 2013), Vegan (v 2.6-4) (Oksanen et al., 2022) and MicroEco (v. 1.9.1) (Liu et al., 2021).

For alpha-diversity, the full dataset was used to calculate Observed, Shannon and Pielou indices and differences between groups were calculated by using a Kruskal-Wallis rank sum test.

For beta-diversity, the ASVs counts were preliminary normalized. Briefly, after visual inspection of rarefaction curves, library were sub-sampled with the "rarefy_even_depth" function (rngseed = 711) from the package phyloseq (v 1.40.0) (McMurdie and Holmes, 2013). Rarefaction is currently the best approach to control for uneven sequencing depth (Schloss, 2024).

Principal Coordinates Analysis (PCoA) were computed to represent dissimilarities between the samples using the Bray-Curtis distance matrix. Differences between groups were assessed by statistical analyses (Permutational Multivariate Analysis of Variance) using the adonis2 function implemented in the vegan package (2.6-4) (Oksanen et al., 2022).

## 2.9. Detection of administered bacteria in 16S barcoding dataset

In order to search for the specific presence of the administered bacteria, we first produced full-length 16S DNA for the identification of the selected candidate bacteria (Dantan et al., 2024a). In parallel, a mock community, created for this study and composed of equal amounts of DNA from four of the administered bacteria (*Bacillus* sp. ARG61, *Vibrio* sp. LTB1, *Halomonas* sp. LTB66, and *Cytobacillus* sp. ARC29) was also submitted to 16S amplicon sequencing in order to validate our method. We then aligned 16S reference sequences of the administered bacteria against all the ASV sequences from the dataset using BLAST (Altschul et al., 1990). ASVs sequences showing percentage of identity greater than 99 % along the full V3V4 marker sequence were considered as our administered bacteria.

# 3. Results

## 3.1. 78 bacterial strains displayed an antibacterial activity

We previously isolated 334 bacteria from the microbiota of *M. gigas* oysters from four different geographical sites (Brest Bay, La Tremblade in Marennes-Oleron Bay, Arcachon Bay and Thau Lagoon) (Dantan et al., 2024a). This collection was screened for their antibacterial activity against eight target bacteria (**Supplementarry_File_1_Table S1**). Among the 334 bacteria from the collection, 78 strains showed an inhibition area around bacterial colony. Among these strains, 32 showed

antibacterial activity against Vibrios (17 against *V. harveyi* Th15_O_A01, 14 against *V. aestuarianus* 02/041, 12 against *V. coralliilyticus* 06/210 and 8 against *V. crassostreae* J2-9) and 65 against opportunistic bacteria associated with POMS disease (49 against *Marinomonas* sp. 12/107-2 T2, 8 against *Pseudoalteromonas* sp. 09/041-1 T3, 6 against *Amphitrea* sp. 14/114-3 T2, and 2 against *Marinobacterium* sp. 05-091-3 T1) (Fig. 2A).

The 76 bacteria presenting an antibacterial activity by co-culture were further screened for the antibacterial activity of their culture supernatant. Thus, height bacteria conserved their activity in the culture supernatant. Among them, three bacterial strains (*Halomonas* sp. LTB66, *Halomonas* sp. LTB97 and *Vibrio* sp. LTB1) presented antibacterial activities in their supernatant against six of the eight target bacteria, two strains (*Bacillus* sp. ARG61 and *Cytobacillus* sp. ARC29) presented antibacterial activities in their culture supernatant against two of the height target bacteria and the three other strains had antibacterial activities in their supernatant against one of the eight target bacteria. (Fig. 2B).

Based on these results, we selected five bacterial strains for further assay. The bacteria were chosen to ensure representation from each geographic origin in the selected bacteria, preferably with antibacterial activity present in the supernatant culture. Thus, *Bacillus* sp. ARG61 from Brest Bay, *Halomonas* sp. LTB66 and *Vibrio* sp. LTB1 from La Tremblade in Marennes-Oleron Bay, *Cytobacillus* sp. ARC29 from Arcachon Bay and *Yoonia* sp. THAU59 from Thau Lagoon were selected for protection assay during an experimental infection.

Four bacterial strains induced a significant reduction of mortality risk during *V. aestuarianus* infection.

To test if exposure to selected bacteria with an antibacterial activity can induce a protective effect against *V. aestuarianus* infection, adult oysters (exposed or control) were challenged with *V. aestuarianus*.

The first mortalities were observed 96 h post cohabitation with donor oysters (Fig. 3). Compared to control condition (survival<sub>t</sub> =408h = 0.14), a significant increase in survival was observed at t = 408 h post-cohabitation for oyster exposed to bacterial strains *Pseudoalteromonas* sp. hCg-42 (survival<sub>408h</sub> = 0.60; *p*-value <0.001), *Yoonia* sp. THAU59 (survival<sub>408h</sub> = 0.49; *p*-value <0.001), *Bacillus* sp. ARG61 (survival<sub>408h</sub> = 0.47; *p*-value = 0.004), and *Cytobacillus* sp. ARC29 (survival<sub>408h</sub> = 0.40; *p*-value = 0.031). No difference in survival was observed for oyster exposed to *Halomonas* sp. LTB66 (survival<sub>408h</sub> = 0.19; *p*-value = 1) or *Vibrio* sp. LTB1 (survival<sub>408h</sub> = 0.07; *p*-value = 1) (Fig. 3)(Table 1). A forest plot analysis confirmed these results and indicates that a significant reduction of the mortality risk of 70 % (Log-Rank test: *p*-value <0.001), 54 % (Log-Rank test: *p*-value <0.001), 46 % (Log-Rank test: *p*-value = 0.002) and 58 % (Log-Rank test: *p*-value <0.01) was observed for the adult oysters exposed to the bacterial strains *Pseudoalteromonas* sp. hCg-42, *Bacillus* sp. ARG61, *Cytobacillus* sp. ARC29 and *Yoonia* sp. THAU59 respectively (**Supplementary_File_2 Fig. S1**).

## 3.2. Addition of beneficial strains slightly modifies the bacterial alpha diversity of the oyster microbiota

To test the immediate effect of the bacterial exposure on oyster microbiota, we analysed the bacterial communities by 16S rRNA gene sequencing after seven days of bacterial exposure with the last addition of bacteria performed 72 h before sampling (Fig. 1).

Sequencing of the V3-V4 hypervariable region of the 16S rRNA gene resulted in a total of 1,713,283 clusters for a total of 40 samples. After a quality control (deleting primers and low-quality sequences, merging, and removing chimeras) and ASV clustering, 1,268,467 reads (74 %) with an average of 31,712 reads per sample were retained for downstream analyses.

Analysis of the alpha diversity metrics (richness, Shannon and Pielou) (Fig. 4) indicates that there are no significant differences for the species richness between the different conditions of bacterial exposure (Fig. 4A). However, we observed a greater range of diversity in the oyster microbiota from the control (217-502), and those exposed to
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Antibacterial activities of bacteria isolated from oysters against the eight target bacteria. The plot represents all the bacteria of the collection having an antibacterial activity against the different target bacteria (A) by coculture and (B) in the culture supernatant. Positive antibacterial activity tests are represented by green tiles. Bacteria written in red (A) are those for which activity was found in the culture supernatants (B). Bacteria selected for experimental infection assay are indicated by a blue triangle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

*Pseudoalteromonas* sp. hCg-42 (82–570) and to *Bacillus* sp. ARG61 (94–590) compared to *Halomonas* sp. LTB66 (289–429), *Cytobacillus* sp. ARC29 (262–595), *Yoonia* sp. THAU59 (267–535) or *Vibrio* sp. LTB1 (183–414) exposure. The alpha diversity with Shannon index (Fig. 4B), which considers taxon diversity and abundance, coupled with the Pielou index (Fig. 4C) which considers the distribution of individuals within species, show trends (NS Kruskal-Wallis test) where oysters exposed to *Cytobacillus* sp. ARC29 (Shannon: 4.35–5.08; Pielou: 0.77–0.81) and

*Yoonia* sp. THAU59 (Shannon: 4.29–5.47; Pielou: 0.77–0.87) have more diverse bacterial communities with a more equitable distribution of species compared to the control condition (Shannon: 3.65–4.88; Pielou: 0.68–0.80) or the condition exposed to *Pseudoalteromonas* sp. hCg-42 (Shannon: 2.86–5.22; Pielou: 0.64–0.82).
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Four bacterial strains have improved oyster survival against *V. aestuarianus* infection. Kaplan-Meier curve representing survival probability of oysters for the control (green solid line) or exposed to candidate bacteria conditions during *Vibrio aestuarianus* infection. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

<table><caption>**Table 1**

Oyster survival data at t = 408 h post *V. aestuarianus* infection.</caption><thead><tr><th rowspan="2">Condition</th><th colspan="3">Survival (at t = 408 h)</th><th colspan="3">Hazard-Ratio (at t = 408 h)</th></tr><tr><th>survival</th><th>std.err</th><th>p-value</th><th>H-R</th><th>95 % CI</th><th>p-value</th></tr></thead><tbody><tr><td>Control</td><td>0.143</td><td>0.0418</td><td>NA</td><td>Reference</td><td>/</td><td>/</td></tr><tr><td><i>Pseudoalteromonas</i> sp. hCg-42</td><td>0.600</td><td>0.0586</td><td>0.000001</td><td>0.30</td><td>0.19-0.48</td><td>&lt;0.001</td></tr><tr><td><i>Bacillus</i> sp. ARG61</td><td>0.471</td><td>0.0597</td><td>0.00398</td><td>0.46</td><td>0.30-0.69</td><td>&lt;0.001</td></tr><tr><td><i>Halomonas</i> sp. LTB66</td><td>0.186</td><td>0.0465</td><td>1.00000</td><td>0.95</td><td>0.66-1.36</td><td>0.778</td></tr><tr><td><i>Cytobacillus</i> sp. ARC29</td><td>0.400</td><td>0.0586</td><td>0.03107</td><td>0.54</td><td>0.36-0.80</td><td>0.002</td></tr><tr><td><i>Yoonia</i> sp. THAU59</td><td>0.486</td><td>0.0597</td><td>0.00032</td><td>0.42</td><td>0.28-0.63</td><td>&lt;0.001</td></tr><tr><td><i>Vibrio</i> sp. LTB1</td><td>0.0714</td><td>0.0308</td><td>1.00000</td><td>1.19</td><td>0.84-1.69</td><td>0.340</td></tr></tbody></table>

### 3.3. Beneficial bacteria do not maintain in oyster tissues but significantly impact the beta diversity of the bacterial microbiota

After 7 days of exposure to the beneficial bacteria, we checked for their presence with the last addition of bacteria performed 72 h before sampling. Doing a blast search against all the ASVs obtained using the 16S DNA sequences of the selected beneficial bacteria as a query, we were not able to detect the administered bacteria in the microbiota of oysters (Supplementary_File_2 Fig. S2A) whereas the four bacteria were detected in the mock community although *Cytobacillus* and *Bacillus* were both affiliated to *Bacillus* (Supplementary_File_2_Figure_S2A). The ASVs from the Mock were thus manually reassigned to their bacterial strain in accordance with the BLAST results in particular for *Bacillus* sp. ARG61 and *Cytobacillus* sp. ARC29 (Supplementary_File_2_Figure_S2B).

Dissimilarity analysis, based on the Bray-Curtis index, on adult oyster microbiota revealed significant differences in microbiota composition between control oysters and oysters exposed to *Pseudoalteromonas* sp. hCg-42, *Bacillus* sp. ARG61, *Halomonas* sp. LTB66,

*Cytobacillus* sp. ARC29, *Yoonia* sp. THAU59 and *Vibrio* sp. LTB1 (Table 2).

At the phylum level, control oysters were dominated by Proteobacteria (54.1 %) and Bacteroidota (23.2 %) followed by Verrucomicrobiota (9.2 %) and Firmicutes (3.6 %) (Fig. 5A). For oysters exposed to selected bacteria, Proteobacteria and Bacteroidota remained the dominant phyla. In comparison with the control condition, oysters exposed to *Pseudoalteromonas* sp. hCg-42 have a greater proportion of Firmicutes (13 %) and Fusobacteriota (12.9 %). The other conditions displayed a relatively similar composition compared to control condition (Supplementary_File_2 Fig. S3). At the Genus level, in the control condition, the most abundant taxa were *Psychromonas* (10.3 %), *Alkalimarinus* (8.6 %), *Persicirhabdus* (7.2 %) and *Vibrio* (5.8 %) (Fig. 5). In comparison, for oysters exposed to *Bacillus* sp. ARG61, *Halomonas* sp. LTB66 and *Cytobacillus* sp. ARC28 the most abundant genera were *Nautella* (19.5, 16.8 and 7.7 % respectively), *Alkalimarinus* (6.5, 3.5 and 3.3 %), *Psychrilyobacter* (5.9, 5.1 and 2.9 %) and *Vibrio* (6.3, 5.6 and 2.9 %). Oysters exposed to *Pseudoalteromonas* sp. hCg-42 displayed a higher proportion of *Psychrilyobacter* (10.8 %) and *Mycoplasma* (4.6 %). Oysters exposed to
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Seven days of bacterial exposure did not induce significant changes in alpha diversity. Boxplot representing the alpha diversity metrics (y axis) of oyster microbiota for control and exposed to selected bacteria (n = 5 oyster per conditions) with Observed (A), Shannon (B) and Pielou (C) indices.</figcaption></figure>

<caption>**Table 2**</caption>

The beneficial bacterial exposure induces significant changes in the bacterial composition of the exposed oysters.

<table><thead><tr><th>Conditions<br>(Compared to control)</th><th>Dum Sq</th><th>R<sup>2</sup></th><th>F</th><th>p-value</th></tr></thead><tbody><tr><td><i>Pseudoalteromonas</i> sp. hCg-42</td><td>0.32</td><td>0.23</td><td>2.09</td><td>0.008</td></tr><tr><td><i>Bacillus</i> sp. ARG61</td><td>0.56</td><td>0.32</td><td>3.81</td><td>0.011</td></tr><tr><td><i>Halomonas</i> sp. LTB66</td><td>0.56</td><td>0.35</td><td>4.23</td><td>0.013</td></tr><tr><td><i>Cytobacillus</i> sp. ARC29</td><td>0.52</td><td>0.32</td><td>3.80</td><td>0.006</td></tr><tr><td><i>Yoonia</i> sp. THAU59</td><td>0.35</td><td>0.20</td><td>2.04</td><td>0.008</td></tr><tr><td><i>Vibrio</i> sp. LTB1</td><td>0.27</td><td>0.19</td><td>1.90</td><td>0.050</td></tr></tbody></table>

Note: analyses were carried out on five oysters per condition, excepted for Pseudoalteromonas sp. hCg-42, for which four oysters were used due to an abnormal sample that was discarded from the analysis.

Results of permanova on the Bray-Curtiss dissimilarity matrix showing the effects of microbial exposure on microbiota community composition compared to the control condition. The p-values were obtained using 100,000 permutations.

Yoonia sp. THAU59 displayed *Mycoplasma* (7.2 %), *Alkalimarinus* (5.4 %) and *Psychrilyobacter* (5 %) as most abundant genera. Last for oysters exposed to *Vibrio* sp. LTB1 we identified *Alkalimarinus* (8.3 %), *Persicirhabdus* (5.8 %), *Vibrio* (5.7 %) *Tenacibaculum* (5.6 %) and

*Psychromonas* (5.5 %) (Fig. 5).

## 4. Discussion

Infectious diseases are a threat for oyster farming, and among them, the pathogenic bacterium *V. aestuarianus* has been observed to spread across Europe (Mesnil et al., 2022). To ensure the sustainability of oyster farming, it is crucial to develop effective, sustainable and socially accepted strategies to fight vibriosis. In this study, we explored the antimicrobial potential of bacteria isolated from oysters, with the aim of identifying natural antagonists that could mitigate the harmful effects of *V. aestuarianus* infections in oyster farms.

Oyster microbiota is a promising source of bacteria with antibacterial activity.

By screening a collection of 334 bacteria, we identified 76 bacteria (22.7 % of the collection) that displayed an antibacterial activity by co-culture, and eight of them (2.4 % of the collection) displaying an antibacterial activity in their supernatant against at least one of the target bacteria. We acknowledge that we may have lost heat sensitive antimicrobial molecules since our protocol to collect the supernatant includes a heating step at 100 °C. This could explain the small proportion of effective molecules present in the supernatant fractions. Furthermore,
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Bacterial composition differs according to bacterial exposure.
Donut plot representing the mean relative abundance of bacterial communities for the control (n = 5) or exposed to selected bacteria (n = 5/condition) oyster samples at the genus level.</figcaption></figure>

the spectrum of activity of the selected bacteria differed depending on the type of antimicrobial tests, either using coculture assay or using the culture supernatant. This could be explained by the loss of certain antibacterial compounds during the centrifugation and/or the heating steps. It also underlines that the methodology used for the assay is a key issue for antimicrobial screening. Further tests using a different protocol for supernatant extraction will therefore be necessary to be as exhaustive as possible. Furthermore, it will be interesting to carry out additional antibacterial activity tests using a broader range of pathogenic targets affecting species of aquaculture interest.

Exposure to four bacterial strains (*Pseudoalteromonas* sp. hCg-42; *Bacillus* sp. ARG61; *Cytobacillus* sp. ARC29 and *Yoonia* sp. THAU59) induced a significant reduction of the mortality risk against *V. aestuarianus* infection.

Four bacterial strains (*Pseudoalteromonas* sp. hCg-42; *Bacillus* sp. ARG61; *Cytobacillus* sp. ARC29 and *Yoonia* sp. THAU59) among 6 tested induced a significant reduction, from 46% to 70%, of the mortality risk against *V. aestuarianus* infection. To our knowledge, this is the first demonstration of a protective effect against *V. aestuarianus* by a potential antagonistic bacterium. The Vibrio challenge was performed 72 h after the last addition of the beneficial bacteria. Longer term effect needs to be tested as the dynamics of Vibrio infection can be long and/or chronic-like (Travers et al., 2017). It is therefore possible that the protection we observed only delayed the progression of the infection. Probiotics have various modes of action that have been the subject of numerous studies in marine farmed species. Their effects against pathogens may result from direct actions, such as competition with pathogens through antibacterial activities in shellfish (Lim et al., 2011; Offret et al., 2018) and fish (Touraki et al., 2012), or quorum quenching

activities in seabass (Ghanei-Motlagh et al., 2021). They may also act indirectly, such as by stimulating the host's immune system as shown in shrimp (Hao et al., 2014; Tseng et al., 2009), in seabream (Guzmán-Villanueva et al., 2014), or in sea cucumber (Yan et al., 2014). Probiotics can also be used to improve nutrient assimilation or host growth in oysters (Aguilar-Macías et al., 2010; Wang et al., 2019). Finally, some probiotics are used to control the chemical and/or microbiological quality of the rearing water in aquaculture facilities (Ma et al., 2009).

Further analyses will be required to determine the mechanisms underlying resistance to *V. aestuarianus*. We did not detect the administered bacteria in the microbiota 72 h after addition. This suggests that the added bacteria are likely not colonising the microbiota as dominant members. Our methodology was validated using a mock assay; however, we cannot rule out the possibility that the added strain may be present in a small amount, which we failed to detect due to a lack of sensitivity. To address this limitation, future investigations should consider the development of specific primers and use of targeted qPCR or droplet digital PCR, which may provide the sensitivity necessary to track low-level colonisation and persistence of administered strains. Furthermore, several studies reported that added probiotic strains do not maintain in their host microbiota. This can be observed in the European abalone *Haliotis tuberculata* where exposure to the *Pseudoalteromonas* sp. hCg-6 exogenous strain resulted in only a temporary presence of the probiotic strain in the haemolymph rather than the establishment of a long-term interaction (Offret et al., 2018). Exposure of *M. gigas* larvae to bacteriocin-like inhibitory substance (BLIS)-producing *Aeromonas*. showed that the probiotic strain concentration decreased right after addition and was not detectable 72 h after exposure (Gibson et al., 1998). Furthermore, among the four strains conferring a protective
effect against *V. aestuarianus*, three bacteria (*Bacillus* sp. ARG61; *Cyto-bacillus* sp. ARC29 and *Yoonia* sp. THAU59) had no antibacterial activity against *V. aestuarianus in vitro*, making the explanation of resistance acquisition even more complex. Taken together, these results suggest that a direct antagonistic effect of the beneficial strains against *Vibrio* may likely not be at the origin of the resistance that we observe in our experiments. The change in the microbiota composition could rather explain the observed beneficial effect. Such an impact on microbiota composition after exposure to beneficial strains has been reported in other species. This beneficial shift in microbiota composition following an exposure to microorganisms has already been observed in Pacific oysters, where exposure to a complete microbiota or mixes of cultivable bacteria led to changes in microbiota coupled with improved survival against POMS infection (Dantan et al., 2024a; Fallet et al., 2022). A similar effect was observed in *Crassostrea virginica* larvae, where exposure to the beneficial marine bacterium *Phaeobacter inhibens* S4 resulted in a restructuring of the larval microbiota, characterised by an increased relative abundance of bacterial taxa belonging to the genera *Alteromonas* and *Pseudomonas*. (Takyi et al., 2024). Exposure of *C. sikamea* oysters to *Streptomyces* strains N7 and RL8 also induced a shift in the diversity and composition of their microbiota, leading to a decrease in *Vibrio* bacteria (García Bernal et al., 2017). An alternative still non-exclusive hypothesis is that the exposure to these bacteria has induced immunostimulation effects as it has been previously reported in oysters, abalone and scallops. Specifically, studies have demonstrated that an exposure of *C. virginica* larvae to probiotic bacteria *Bacillus pumilus* RI06-95 and *Phaeobacter inhibens* S4 led to improved survival in the face of *V. coralliilyticus* RE22 infection simultaneously with an activation of immune signalling pathways (NF-kB and MAPK pathways) and expression of immune effectors such as serine protease inhibitor (Cv-spi2), mucins and antimicrobial histone H2B (Modak and Gomez-Chiarri, 2020). Similarly, an exposure of *M. gigas* larvae to a mix of probiotics bacteria, led to an increased expression of immune signalling proteins (TOLL) and immune effectors such as interleukin IL-17 or MD88. Furthermore, larvae exposed to this mix of probiotics showed an increased survival against *V. coralliilyticus* infection (Hesser et al., 2024). Comparatively, *Argopecten purpuratus* scallop larvae exposed to bacterial strains belonging to *Psychrobacter*, *Hydrogenophaga*, and *Shewanella* genera displayed no mortality after 24 h post infection to *V. bivalvicida* VPAP30. This beneficial effect could be due to an immunomodulation of genes coding for opsonin, superoxide dismutase (SOD), Toll-like receptor (TLR) or Lysozyme following the bacterial exposure (Munoz-Cerro et al., 2024). At last, New Zealand black-footed abalone *Haliotis iris*, displayed a significantly increase in the number of total haemocytes count (HTC), non-apoptotic cells, an higher percentage of ROS-positive cells and a higher viability following an exposure to multi-strain probiotics (Grandiosa et al., 2018). In a broader context, exposure to various abiotic elicitors may also increase immune response by inducing gene expression changes in cephalopod molluscs (Li et al., 2023, 2024; Zhao et al., 2025).

## 5. Conclusion

In this study, we have shown that exposure to bacteria isolated from *M. gigas* microbiota and displaying *in vitro* antibacterial activities against various pathogens or opportunists, could increase oyster survival following experimental infectious challenge with *V. aestuarianus*. Further studies will be needed to understand the immune molecular mechanisms involved in the tolerance conferred by these bacteria. It would also be interesting to characterise and purify molecules that are active against pathogens and opportunists. These molecules may offer a promising way to mitigate the effects of these infectious diseases. A sustainable oyster farming is a key to develop this growing industry in a context of global changes and emerging infectious diseases. The development of prophylactic methods such as the use of probiotics is a necessity.

## CRediT authorship contribution statement

**Luc Dantan:** Writing – original draft, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Marie-Agnès Travers:** Writing – review & editing, Validation, Methodology, Conceptualization. **Lionel Degremont:** Writing – review & editing, Investigation, Conceptualization. **Benjamin Morga:** Writing – review & editing, Investigation, Conceptualization. **Prunelle Carcassonne:** Investigation. **Mickael Mege:** Investigation, Conceptualization. **Yannick Fleury:** Conceptualization. **Bruno Petton:** Methodology, Investigation, Conceptualization. **Elise Maurouard:** Investigation. **Jean-François Allienne:** Investigation. **Gaëlle Courtay:** Investigation. **Océane Romatif:** Investigation. **Raphaël Lami:** Methodology. **Laurent Intertaglia:** Methodology. **Yannick Gueguen:** Project administration, Funding acquisition, Conceptualization. **Jeremie Vidal-Dupiol:** Writing – review & editing, Validation, Project administration, Methodology, Funding acquisition, Conceptualization. **Céline Cosseau:** Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. **Eve Toulza:** Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

## Ethical approval

The animal (oyster *Magallana gigas*) testing followed all European regulations concerning animal experimentation. The authors declare that the use of genetic resources fulfils the French and EU regulations on the Nagoya Protocol on Access and Benefit-Sharing (French legislation 2019-486).

## Fundings

The present study was supported by the Ifremer project GT-huitre and by the Fonds Européen pour les Affaires Maritimes et la Pêche (FEAMP, GESTINNOV project n°PFEA470020FA1000007), the project "Microval" of the Bonus Qualité Recherche program of the University of Perpignan, the project "gigantimic 1" from the Fédération de Recherche Energie Environnement of the University of Perpignan, the project "gigantimic 2" from the Kim Food and Health foundation of MUSE of the University of Montpellier and the project ANR DECICOMP (ANR-19-CE20-0004). This study is set within the framework of the "Laboratoires d'Excellence (LABEX)": TULIP (ANR-10-LABX-41) and CeMEB (ANR-10-LABX-04-01). Luc Dantan is a recipient of a PhD grant from the Region Occitanie (Probiomic project) and the University of Perpignan Via Domitia Graduate School ED305.

## Declaration of competing interest

The authors declare that they have no competing interests.

## Acknowledgements

The authors thank Céline Garcia from Ifremer, EU Reference Laboratory for mollusc diseases (La Tremblade, France) for providing oyster bacterial isolates used as target bacteria. We are grateful to Leo Duperret, Emily Kunselman, Nicole Faury, Cyrielle, Lecadet and Delphine Tourbiez for their help during the oyster experimental infections. We also like to thank Antoine Jourdan and both hatchery and nursery teams at PMMLT La Tremblade and PMM Bouin for the supply and care of the oysters. We are grateful to the BIO2MAR platform (http://bio2mar.obs-banyuls.fr) for access to the instrumentation.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.aquaculture.2025.742949.

## Data availability

Raw sequence data for 16S sequencing for metabarcoding analysis have been made available through the SR A database (BioProject accession number PRJNA1183305).

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