Identification of bacterial pathogens in cultured fish with a custom peptide database constructed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Abstract

Background: The majority of infectious diseases of cultured fish is caused by bacteria. Rapid identification of bacterial pathogens is necessary for immediate management. The present study developed a custom Main Spectra Profile (MSP) database and validate the method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of fish bacterial pathogens. Streptococcus agalactiae, Streptococcus iniae, Aeromonas hydrophila, Aeromonas veronii, and Edwardsiella tarda obtained from diseased fish were used as representative bacterial pathogens in this study. Bacterial peptides were extracted to create a Main Spectra Profile (MSP), and the MSPs of each bacterial species was added into the MALDI Biotyper database. Fifteen additional isolates of each bacterial species were tested to validate the utilized technique.

Results: The MSPs of all field isolates were clearly distinguishable, and the MSPs of the same species were clustered together. The identification methodology was validated with 75 bacterial isolates. The reliability and specificity of the method were determined with MALDI Biotyper log score values and matching results with 16 s rDNA sequencing. The species identification using the public MALDI Biotyper library (Bruker MALDI Biotyper) showed unreliable results (log score < 2.000) with 42.67% matching result with the reference method. In contrast, accurate identification was obtained when using the custom-made database, giving log score > 2.115, and a 100% matching result.

Conclusion: This study demonstrates an effective identification of fish bacterial pathogens when a complete custom-made MSP database is applied. Further applications require a broad, well-established database to accommodate prudent identification of many fish bacterial pathogens by MALDI-TOF MS.

Keywords: Biotyper; Fish disease; Mass spectrometry; Proteomic.

Fig. 1

Mass peptide fingerprints of five bacterial pathogens (S. agalactiae, S. iniae, A. hydrophila, A. veronii and E. tarda) isolated from cultured fish

Fig. 2

Cluster analysis of the five bacterial pathogens (S. agalactiae, S. iniae, A. hydrophila, A. veronii and E. tarda) isolated from cultured fish. a The 3D-PCA scatterplot representing clusters of each species (dashed circles) and the (b) MSP dendrogram of the representative bacterial isolates analyzed in the present study with the reference ATCC strains