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)

Patharapol Piamsomboon,Tran Quang Hung,Janthima Jaresitthikunchai,Sittiruk Roytrakul,Janenuj Wongtavatchai

doi:10.1186/s12917-020-2274-1

Abstract

BackgroundThe 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.ResultsThe 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.ConclusionThis 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.

Highlights

The majority of infectious diseases of cultured fish is caused by bacteria
The three-dimensional principal component analysis (3D-PCA) scatterplot presented clearly distinguishable clusters, each cluster presented in the 3DPCA scatterplot (Fig. 2a) indicates Main Spectra Profile (MSP) or distinctive peptide fingerprint of the bacterial species
Bacterial isolates of the same species were grouped within the same clade of MSP dendrogram (Fig. 2b)

Summary

Introduction

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. Conventional microbiology techniques, including morphological, physiological and biochemical tests, and molecular techniques based on 16S rDNA sequencing, are the gold standard for bacterial species identification [5]. These techniques require a substantial amount of time and expensive reagents [6]. These mass spectra libraries are able to differentiate the bacteria to their genus, species or sub-group levels subject to sufficient pre-existing reference strains in the database [10]

Methods

Results

Conclusion