MALDI-TOF database for the identification of potentially pathogenic Vibrio in marine molluscs
|Author(s)||Moussa Pouly Mirna1, Cauvin Elodie3, Le Piouffle Anthony4, Bidault Adeline6, Paillard Christine6, Benoit Fabienne3, Thuillier Benoît4, Treilles Michaël5, Travers Marie-Agnes1, 2, Garcia Celine1|
|Affiliation(s)||1 : Ifremer, Laboratoire de Génétique et Pathologie des Mollusques Marins, Avenue de Mus de Loup, 17390 La Tremblade, France
2 : IHPE, Université de Montpellier, CNRS, Ifremer, Université de Perpignan Via Domitia, Place E. Bataillon, 34095, Montpellier, France.
3 : Labeo-Manche, 1352 avenue de Paris 50000 Saint-Lô, France
4 : Labocea, Avenue de la Plage des Gueux 29330 Quimper, France
5 : Qualyse, ZI Montplaisir, 79220 Niort, France
6 : Univ Brest, CNRS, IRD, Ifremer, UMR 6539 LEMAR, F-29280 Plouzane, France
|Keyword(s)||Maldi-tof, Vibrio, Marine mollusc pathogens, Vibrio database|
In mollusc aquaculture, a large number of Vibrio species are considered as major pathogens that can cause high losses in hatchery and field. Thus, development of effective techniques for rapid detection and accurate identification of Vibrio involved in mortality events appears important to build efficient mollusc diseases surveillance programs. Phenotypic, biochemical and molecular techniques based on DNA amplification and sequencing are widely used for the identification of Vibrio species in the environment, but are time-consuming because of the use of different markers to differentiate closely related species. To provide the correct identification of unknown bacteria and species classification of environmental isolates, a tool is increasingly used, the matrix-assisted laser desorption/ionization time of flight spectrometry (MALDI-TOF), a proteomic method able to generate a specific proteomic bacteria profile in few seconds. Nevertheless, existing databases do not contain spectra for Vibrio associated with marine molluscs, consequently, we proposed to create a MALDI-TOF VibrioBase database containing containing 120 reference spectra of Vibrio species potentially responsible for molluscs diseases and belonging to 25 species: V. aestuarianus, V. cortegadensis, V. tapetis and species of Coralliilyticus, Harveyi, Mediterranei, Orientalis clades. A total of 72 mass proteomic spectra per strain cultured in three different media were generated and analyzed to determine specific reference spectra for each strain in order to perform the MALDI-TOF MS database specific to marine molluscs.To increase the specificity of the reference spectra, complementary statistical methods (PCA clustering and CCI matrix) were used. A perfect discrimination was obtained with all the created MSP of Vibrio species and even for very close related species.
This VibrioBase is a first step that could enable the use of MALDI-TOF MS as a routine diagnostic tool for rapid bacterial identification in marine molluscs.This new database will be extended by including others strains of marine Vibrio as strains of Splendidus clade.
|Utilisation||The Vibrio MALDI-TOF database is published without any warranty, express or implied. The user assumes all risk arising from his/her use of this database. The Vibrio MALDI-TOF database is intended to be research-quality and includes estimates of data quality and accuracy, but it is possible that these estimates or the data themselves contain errors. It is the sole responsibility of the user to assess if the data are appropriate for his/her use, and to interpret the data, data quality, and data accuracy accordingly. Users of this database could ask questions and report problems to the contact addresses listed in the data files.|
|Acknowledgments||We wish to thank Delphine Tourbiez and Mathilde Noyer for their assistance in bacteriology and molecular analyses. Many thanks are also adressed to Dr Frédérique le Roux and Dr Delphine Destoumieux-Gazon for their assistance and for sharing their Vibrio strains. This study is set within the framework of the "Laboratoires d'Excellences (LABEX)" TULIP (ANR-10-LABX-41). This work was supported by Ifremer; by DGAL through the National Reference Laboratory for Mollusc Diseases, Ifremer, La Tremblade; and by the European Commission through the European Union Reference Laboratory, Ifremer, La Tremblade. This work was also partly supported by the EU-funded project VIVALDI (‘Preventing and Mitigating Farmed Bivalve Diseases’) H2020 program, no. 678589.|
Spectra were acquired using the Microflex LT/SH system (Bruker Daltonics Inc. Bremen). Each spot was measured 3 times, resulting in 24 single spectra per medium for each strain. A total of 72 spectra for each strain were generated.