Establishment and Application of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Detection of Shewanella Genus

Abstract

Shewanella species are widely distributed in the aquatic environment and aquatic organisms. They are opportunistic human pathogens with increasing clinical infections reported in recent years. However, there is a lack of a rapid and accurate method to identify Shewanella species. We evaluated here matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of Shewanella. A peptide mass reference spectra (PMRS) database was constructed for the type strains of 36 Shewanella species. The main spectrum projection (MSP) cluster dendrogram showed that the type strains of Shewanella species can be effectively distinguished according to the different MS fingerprinting. The PMRS database was validated using 125 Shewanella test strains isolated from various sources and periods; 92.8% (n = 116) of the strains were correctly identified at the species level, compared with the results of multilocus sequence analysis (MLSA), which was previously shown to be a method for identifying Shewanella at the species level. The misidentified strains (n = 9) by MALDI-TOF MS involved five species of two groups, i.e., Shewanella algae-Shewanella chilikensis-Shewanella indica and Shewanella seohaensis-Shewanella xiamenensis. We then identified and defined species-specific biomarker peaks of the 36 species using the type strains and validated these selected biomarkers using 125 test strains. Our study demonstrated that MALDI-TOF MS was a reliable and powerful tool for the rapid identification of Shewanella strains at the species level.

Keywords: MALDI-TOF mass spectrometry; Shewanella; detection; establishment and application; multilocus sequence analysis.

FIGURE 1

Dendrogram of the cluster analysis of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra. The scale below the dendrogram represents the degree of difference in the mass spectrometry fingerprinting of the 36 Shewanella type strains, and the difference level reflects the relationship between each other, with the value between 0 and 1. With the main spectrum projection (MSP) similarity of 77.6% as the critical value, 36 type strains of Shewanella were divided into six groups. Among them, Algae, Gelidii, Aquimarina, and Putrefaciens included multiple type strains (species); S. aestuarii and S. hanedai stood separately in their own clade.

FIGURE 2

Phylogenetic tree constructed based on six concatenated gene sequences [gyrA, gyrB, infB, recN, rpoA, and topA (4,191 bp)] of 36 Shewanella type strains and 125 test strains (by the neighbor-joining method). Each black triangle contains the test strains of the same species, and the number of test strains is shown in brackets. Numbers on branches are bootstrap values in percentage from 1000 replicates.

FIGURE 3

Comparison of identification between multilocus sequence analysis (MLSA) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for 125 test strains. Different colors represent different Shewanella species.

FIGURE 4

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra of the five species displayed in the MDT Master: (A) (a) Peak at 10,065 m/z of S. algae compared with S. chilikensis and S. indica; (b) comparison of peak at 3,307 m/z belonging to S. chilikensis to S. algae, S. indica, respectively. (c) Different distribution of peak at 4,827 m/z belonging S. indica compared with S. algae and S. chilikensis. (B) Different distribution of peaks at 4,221, 3,778, and 9,574 m/z between S. xiamenensis and S. seohaensis.