A MALDI-TOF MS database for fast identification of Vibrio spp. potentially pathogenic to marine mollusks

Abstract

In mollusk aquaculture, a large number of Vibrio species are considered major pathogens. Conventional methods based on DNA amplification and sequencing used to accurately identify Vibrio species are unsuitable for monitoring programs because they are time-consuming and expensive. The aim of this study was, therefore, to develop the MALDI-TOF MS method in order to establish a rapid identification technique for a large panel of Vibrio species. We created the EnviBase containing 120 main spectra projections (MSP) of the Vibrio species that are potentially responsible for mollusk diseases, comprising 25 species: V. aestuarianus, V. cortegadensis, V. tapetis and species belonging to the Coralliilyticus, Harveyi, Mediterranei, and Orientalis clades. Each MSP was constructed by the merger of raw spectra obtained from three different media and generated by three collaborating laboratories to increase the diversity of the conditions and thus obtain a good technique robustness. Perfect discrimination was obtained with all of the MSP created for the Vibrio species and even for very closely related species as V. europaeus and V. bivalvicida. The new EnviBase library was validated through a blind test on 100 Vibrio strains performed by our three collaborators who used the direct transfer and protein extraction methods. The majority of the Vibrio strains were successfully identified with the newly created EnviBase by the three laboratories for both protocol methods. This study documents the first development of a freely accessible database exclusively devoted to Vibrio found in marine environments, taking into account the high diversity of this genus. KEY POINTS: • Development of a MALDI-TOF MS database to quickly affiliate Vibrio species. • Increase of the reactivity when faced with Vibrio associated with mollusk diseases. • Validation of MALDI-TOF MS as routine diagnostic tool.

Keywords: Database; MALDI-TOF MS; Marine mollusk pathogens; Vibrio.

Fig. 1

Main steps of EnviBase creation (a) and validation (b). ¹Free download on 10.17882/75416. ²Erler et al. , available on demand. ³Need subscription for downloading and annual updates (Bruker Daltonics, Inc.)

Fig. 2

Raw spectral analysis using three different methods on the strain Vibrio bivalvicida CECT 8856. a PCA cluster analysis. Classification of the proteomic mass spectra of the strain cultivated in three different media in the first three principal components (PC1, PC2, PC3) using MALDI Biotyper Compass Explorer software. 1x and 1y: 1.5% salted TSA medium. 2: Zobell medium. 3: marine agar medium. b Raw spectra. Visualization of the spectra using the flexAnalysis software. c CCI matrix. CCI values obtained using spectra from group 1. Cold colors indicate weakly correlated spectra, and warm colors indicate highly correlated spectra

Fig. 3

Dendrogram generated using Bruker’s MBT Compass Explorer based on MSP spectra. Species affiliations are indicated by the colors. Genetically defined clade affiliations are also indicated on the right-hand side

Fig. 4

Identification of the new main reference spectra (MSP) with the Bruker^TM database and Erler’s database. a Only the MSP of the Vibrio species present in both databases were tested. b Vibrio species not present in either database. The highest matching scores of the new main reference spectra were represented by ranges indicating highly probable species identifications (≥2.3), probable species identifications (2.0–2.299), secure genus identifications (1.7–1.999), and unreliable identifications (<1.7). Assignment of strains obtained with Bruker^TM database that were different from the assignment of species of EnviBase main spectra were classified as “different identification”

Fig. 5

Blind test on the Vibrio sp. present in the database identified by MALDI-TOF MS with the new created EnviBase performed by one laboratory using the (a) direct transfer and (b) protein extract methods. Different colors are used to indicate the score values

Fig. 6

MALDI-TOF MS microbial identification workflow. ¹Free download on 10.17882/75416. ²Erler et al. , available on demand. ³Need subscription for downloading and annual updates (Bruker Daltonics, Inc.)