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Zebrafish gut colonization by mCherry-labelled lactic acid bacteria

  • Applied genetics and molecular biotechnology
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Abstract

A critical feature of probiotic microorganisms is their ability to colonize the intestine of the host. Although the microbial potential to adhere to the human gut lumen has been investigated in in vitro models, there is still much to discover about their in vivo behaviour. Zebrafish is a vertebrate model that is being widely used to investigate various biological processes shared with humans. In this work, we report on the use of the zebrafish model to investigate the in vivo colonization ability of previously characterized probiotic lactic acid bacteria. Lactobacillus plantarum Lp90, L. plantarum B2 and Lactobacillus fermentum PBCC11.5 were fluorescently tagged by transfer of the pRCR12 plasmid, which encodes the mCherry protein and which was constructed in this work. The recombinant bacteria were used to infect germ-free zebrafish larvae. After removal of bacteria, the colonization ability of the strains was monitored until 3 days post-infection by using a fluorescence stereomicroscope. The results indicated differential adhesion capabilities among the strains. Interestingly, a displacement of bacteria from the medium to the posterior intestinal tract was observed as a function of time that suggested a transient colonization by probiotics. Based on fluorescence observation, L. plantarum strains exhibited a more robust adhesion capability. In conclusion, the use of pRCR12 plasmid for labelling Lactobacillus strains provides a powerful and very efficient tool to monitor the in vivo colonization in zebrafish larvae and to investigate the adhesion ability of probiotic microorganisms.

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Acknowledgments

We sincerely thank Dr. César Martín Plágaro (UPV/EHU, Leioa, Spain) for confocal microscopy analysis. We thank Dr. Stephen Elson for the critical reading of the manuscript.

The work performed in Spain was supported at the Centro de Investigaciones Biológicas by grant AGL2012-40084-C03-01 from the Spanish Ministry of Economics and Competitiveness and at AZTI-Tecnalia by the “Departamento de Desarrollo Económico y Competitividad Viceconsergería de Agricultura, Pesca y Politicas Alimentarias” (Project FOODBASK3). Iñaki Iturria is a recipient of a PhD fellowship from Iñaki Goenaga Foundation.

The work performed in Italy was supported by the Italian Ministry for Development in the framework of the “Industria 2015 Bando Nuove Tecnologie per il Made in Italy—Realizzazione di una innovativa pasta alimentare funzionale arricchita di componenti bioattivi e probiotici”, and by MIUR (PON02_00186_2937475) in the framework of the project named “Protocolli innovativi per lo sviluppo di alimenti funzionali” “(Pro.Ali.Fun.)”.

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    Authors and Affiliations

    1. Department of Agriculture, Food and Environment Sciences, University of Foggia, via Napoli 25, 71122, Foggia, Italy

      Pasquale Russo, Graziano Caggianiello & Giuseppe Spano

    2. Food Research Unit, Parque Tecnológico de Bizkaia Astondo Bidea, Edif. 609 Derio, Bizkaia, Spain

      Iñaki Iturria, Sandra Rainieri & Miguel Angel Pardo

    3. Department of Molecular Microbiology and Infection Biology, Centro de Investigaciones Biológicas (CIB), Madrid, Spain

      Maria Luz Mohedano, Graziano Caggianiello & Paloma López

    4. Department of Clinical and Experimental Medicine, via Pinto, Foggia, Italy

      Daniela Fiocco

    5. Promis Biotech srl, via Napoli 25, 71122, Foggia, Italy

      Pasquale Russo

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    1. Pasquale Russo
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    Correspondence to Giuseppe Spano.

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    Russo Pasquale and Iturria Iñaki contributed equally to this work.

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    Russo, P., Iturria, I., Mohedano, M.L. et al. Zebrafish gut colonization by mCherry-labelled lactic acid bacteria. Appl Microbiol Biotechnol 99, 3479–3490 (2015). https://doi.org/10.1007/s00253-014-6351-x

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