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Potential of Marine Strains of Pseudoalteromonas to Improve Resistance of Juvenile Sea Bass to Pathogens and Limit Biofilm Development

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Abstract

The European sea bass (Dicentrarchus labrax), one of the most produced marine fish species in Europe, is acutely vulnerable to multiple infectious hazards. In this study, we investigated the potential probiotic effect of some marine Pseudoalteromonas bacterial strains against two major pathogens of this species, Vibrio harveyi and the nervous necrosis virus (NNV), and examined their antibiofilm effect. Impregnation phase was done by repeated immersion of juvenile’s sea bass during 8 to 12 weeks in seawater containing the probiotic candidates at a concentration of 106 CFU/mL. Four candidates were tested: (1) a combination of two strains producing antimicrobial compounds, hCg-42 and hOe-125; (2) strain 3J6, with known antibiofilm properties; (3) strain RA15, from the same genus, but with no identified probiotic effect; and (4) a control group without probiotics. At the end of the impregnation phase, fish underwent an infection challenge with V. harveyi or with a pathogenic strain of NNV and mortality was monitored. For the V. harveyi challenge, improved survival rates of 10 and 25% were obtained for the RA15 and the mix hCg-42 + hOe-125-impregnated groups, respectively. For the NNV challenge, no significant benefic effect of the probiotics on infection kinetics or cumulative mortality was observed. At the end of the impregnation phase, the maximal thickness of biofilm was significantly lower in the 3J6, double strain, and RA15 groups, compared with the non-impregnated control group. This study highlights the interesting probiotic potential of marine bacteria to limit mortalities induced by bacterial pathogens as well as biofilm development.

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Data Availability

The sequences of the strains have been published in the studies cited in the bibliography. All data obtained are presented in the article or in the Supplementary Data.

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Acknowledgements

We warmly thank Dr. Alain Le Breton (VetEau, Grenade-sur-Garonne, France) for providing the pathogenic strain of Vibrio harveyi used in this study.

Funding

This work was funded by the European Maritime, Fisheries, and Aquaculture Fund (EMFAF), measure 47, under the name “Projet PaqMan: Développement de probiotiques en Aquaculture Marine”, OSIRIS number PFEA470019FA1000012.

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Author notes

  1. M. Danion and T. Morin shared last authors.

Authors and Affiliations

  1. Virology, Immunology and Ecotoxicology of Fish Unit, ANSES, Ploufragan-Plouzané-Niort Laboratory, National Reference Laboratory for Regulated Fish Diseases, National Research Infrastructure Emerg’In, 29280, Plouzané, France

    A. Rahmani, M. Baud, E. Debosse, J. Cabon, L. Louboutin, M. Danion & T. Morin

  2. Laboratoire de Biotechnologie et Chimie Marines, Université Bretagne Sud, EMR CNRS 6076, IUEM, 29000, Quimper, France

    L. Parizadeh, Y. Fleury & H. Cuny

  3. Laboratoire LIttoral ENvironnement et Sociétés (LIENSs), UMR 7266, CNRS - La Rochelle Université, 17000, La Rochelle, France

    L. Parizadeh

  4. SYSAAF, Station LPGP/INRAE, Campus de Beaulieu, 35042, Rennes, France

    Y. Francois

  5. Laboratoire de Biotechnologie et Chimie Marines, Université Bretagne Sud, EMR CNRS 6076, IUEM, 56100, Lorient, France

    A. Bazire & S. Rodrigues

  6. UMR 5805, Université de Bordeaux, CNRS, Bordeaux INP, EPOC, 33600, Pessac, France

    L. Bellec

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Contributions

TM, YFl, and MD acquired funds and coordinated the study. AR performed all experimental assays, the production of protective strains, and the analyses of the samples harvested with advice of TM, MD, MB, YFr, ED, and LP and LB. LP and YFl performed in vitro inhibition tests on V. harveyi. HC performed primers design for qPCR. AB, SR, and AR performed biofilm measurement by microscopy. JC and LL produced the viral suspension for the NNV challenge. AR, TM, and MD wrote the manuscript (first draft was written by AR). The manuscript was carefully reviewed by other co-authors, who all approved the final version.

Corresponding author

Correspondence to T. Morin.

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All animal experiments were approved by the French Ministry of National Education, Higher Education and Research under the authorization number APAFIS #32741–2020121509556347 v5.

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The authors declare no competing interests.

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The original version of this article has been revised to correct the affiliation of the 6th author, S. Rodrigues.

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Fig.S1

In vitro antibacterial activity of different Pseudoalteromonas strains against Vibrio harveyi 94473 1811603 AQN553P2. Presence of antibacterial activity (i.e. inhibition halo around agar punch holes) induced by Pseudoalteromonas hCg-42, Pseudoalteromonas hCg-6 (type strain) against V. harveyi used in this study. PMB: Polymyxin B (positive control against Gram-negative target bacteria), MB: Marine Broth (negative control). Pseudoalteromonas RA15 was used as a negative control strain due to its lack of antibacterial activity. file1 (TIF 159 KB)

Fig.S2

Cell mortality measured by flow cytometry 15 days post-infection with Vibrio harveyi (Batch 1). Analyses were done for each group, i.e. non-infected control, infected control, RA15, combined strains hCg-42+hOe-125 and 3J6. Dead cell counts were calculated based on the measure of propidium iodide (PI) fluorescence. Three subpopulations were described based on size (FCS) and complexity (SSC) parameters, defined during flow cytometry acquisition. (TIF 362 KB)

Fig.S3

Percentage of phagocytosis activity, measured by flow cytometry, at 15 days post-infection with V. harveyi (Batch 1). Analyses were done in each treatment group, i.e. non-infected control, infected control, RA15, combined strains hCg-42+hOe-125 and 3J6. Phagocytosis percentage was determined based on green fluorescence of internalized fluorescent beads. Measured was performed on the monocyte and granulocyte populations, after exclusion of the region corresponding to non-internalized fluorescent beads. (TIF 255 KB)

Fig.S4

Cell mortality measured by flow cytometry at 12 weeks of impregnation (i.e. just before infection), at 96 h post-infection and at 7 days post-infection with the nervous necrosis virus (NNV) (Batch 2). Analyses were done in each treatment group, i.e. non-infected (NI) control, infected control, RA15, combined strains hCg-42+hOe-125 and 3J6. Dead cells counts were calculated based on the measure of propidium iodide (PI) fluorescence. Two subpopulations were described based on size (FCS) and complexity (SSC) parameters, defined during flow cytometry acquisition. This graph gives the percentage of dead cells in the lymphocyte population. Different letters indicate represent statistical differences (p < 0.05). ns: non-significant. (TIF 381 KB)

Fig.S5

Cell mortality measured by flow cytometry at 12 weeks of impregnation (i.e. just before infection), at 96 h post-infection and at 7 days post-infection with the nervous necrosis virus (Batch 2). Analyses were done in each treatment group , i.e. non-infected (NI) control, infected control, RA15, combined strains hCg-42+hOe-125 and 3J6. Dead cells counts were calculated based on the measure of propidium iodide (PI) fluorescence. Two subpopulations were described based on size (FCS) and complexity (SSC) parameters, defined during flow cytometry acquisition. This graph gives the percentage of dead cells in the monocyte and granulocyte populations. ns: non-significant. (TIF 370 KB)

Fig.S6

Percentage of phagocytosis activity, at 12 weeks of impregnation (i.e. just before infection), at 96 h post-infection and at 7 days post-infection with the nervous necrosis virus (NNV) (Batch 2). Analyses were done in each treatment group: i.e. non-infected (NI) control, infected control, RA15, combined strains hCg-42+hOe-125 and 3J6. Phagocytosis percentage was determined based on green fluorescence of internalized fluorescent beads. Measured was performed on monocytes and granulocytes population, after exclusion of the region corresponding to non-internalized fluorescent beads. Different letters indicate statistical differences (p < 0.05). ns: non-significant. (TIF 344 KB)

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Rahmani, A., Parizadeh, L., Baud, M. et al. Potential of Marine Strains of Pseudoalteromonas to Improve Resistance of Juvenile Sea Bass to Pathogens and Limit Biofilm Development. Probiotics & Antimicro. Prot. (2023). https://doi.org/10.1007/s12602-023-10180-5

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