Rapid classification and identification of salmonellae at the species and subspecies levels by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry

Abstract

Variations in the mass spectral profiles of multiple housekeeping proteins of 126 strains representing Salmonella enterica subsp. enterica (subspecies I), S. enterica subsp. salamae (subspecies II), S. enterica subsp. arizonae (subspecies IIIa), S. enterica subsp. diarizonae (subspecies IIIb), S. enterica subsp. houtenae (subspecies IV), and S. enterica subsp. indica (subspecies VI), and Salmonella bongori were analyzed to obtain a phylogenetic classification of salmonellae based on whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometric bacterial typing. Sinapinic acid produced highly informative spectra containing a large number of biomarkers and covering a wide molecular mass range (2,000 to 40,000 Da). Genus-, species-, and subspecies-identifying biomarker ions were assigned on the basis of available genome sequence data for Salmonella, and more than 200 biomarker peaks, which corresponded mainly to abundant and highly basic ribosomal or nucleic acid binding proteins, were selected. A detailed comparative analysis of the biomarker profiles of Salmonella strains revealed sequence variations corresponding to single or multiple amino acid changes in multiple housekeeping proteins. The resulting mass spectrometry-based bacterial classification was very comparable to the results of DNA sequence-based methods. A rapid protocol that allowed identification of Salmonella subspecies in minutes was established.

FIG. 1.

MALDI-TOF MS profiles and dendrogram of S. enterica subsp. arizonae (Arizonae) and S. enterica subsp. houtenae (Houtenae) grown on different agar media. Duplicate spectra for representative samples are shown, as visualized using a gel view representation. The signal intensities of the peaks were translated into grayscale using BioNumerics 5.1 (Applied Maths). PCA, plate count agar; SBA, sheep blood agar; MHA, Mueller-Hinton agar; MHB, Mueller-Hinton blood agar.

FIG. 2.

Overlay of MALDI mass spectra for strains of S. bongori (group V) (red), S. enterica subsp. enterica (subspecies I) (gray), S. enterica subsp. salamae (subspecies II) (green), S. enterica subsp. arizonae (subspecies IIIa) (dark blue), S. enterica subsp. diarizonae (subspecies IIIb) (yellow), S. enterica subsp. houtenae (subspecies IV) (black), and S. enterica subsp. indica (subspecies VI) (light blue). The graph shows protein peaks in the molecular mass region from 13,230 to 15,270 Da. Assigned ribosomal proteins and peak specificities are indicated as follows: g genus specific; s, species specific; subspecies numbers, subspecies specific.

FIG. 3.

Salmonella species discrimination: WARD-generated dendrogram obtained using Pearson correlation (BioNumerics 5.1) and gel view of mass data obtained from whole-cell MALDI-TOF MS analyses of S. bongori and S. enterica strains.

FIG. 4.

Phylogenetic classification of Salmonella species and subspecies based on variations in whole-cell MALDI-TOF mass data for multiple protein biomarkers. The dendrogram was calculated based on the binary table extracted from Table S2 in the supplemental material using the simple matching similarity coefficient and complete linkage (BioNumerics 5.1).