Conventional methods for bacterial identification involve multiple enrichment, isolation and biochemical tests, which typically requires 5-7 days. Mass spectrometric analysis has emerged as a promising means for rapid microbial identification. MALDI-TOF MS-based methods have gained much attention due to its speed and simplicity; however, it may not always be applicable for distinguishing closely related bacteria and accurate identification is only achieved to the species level. Indeed, a recent study on three commercially available MALDI-TOF MS-based biotyping systems showed that (1) all three systems fail to differentiate Shigella from E coli. and (2) only one system correctly assigns Salmonella isolates to the species level. However, for most food safety applications a minimum of subspecies or serovar level identification is needed. Here we propose a peptide-based LC-MS/MS method that will provide improved proteome coverage by extending the upper size limit of identifiable proteins and providing identification of proteins that were insoluble by intact analysis. Shigella (Sonnei and Flexneri), E. coli. (O157:H7 and K-12), and Salmonella A collection serovars (Typhimurium, Saintpaul, Heidelberg, Paratyphi B, and Muenchen) were used in this study. Our data showed that ~ 7000 peptides were routinely identified using 100 ng of bacterial lysate from a pure culture. The proteomic analysis reveals hundreds of peptide ions that differentiate the Salmonella strains from the E. coli and Shigella. In contrast, the Shigella and E. coli species share a very high degree of protein homology. The data suggest that Shigella and E. coli. are closely related but quite distinct from Salmonella. Closer inspection of the Shigella and E coli. data reveal tens of peptide ions that are species-specific, which can serve as diagnostic markers. Further, despite the extreme protein homology across the Salmonella serovars, a number of serovar-differentiating peptide markers that are derived from cell surface proteins or proteins containing single or multiple amino acid exchanges were identified. The specificity of these peptide markers were confirmed using BLAST searches against NCBI's non-redundant (nr) protein database. Our data show that sets of peptides are detectable by LC-MS/MS that differentiate closely related Salmonella serovars. This represents a marked improvement in specificity over commercial MALDI-TOF based bacterial ID platforms.

Identification of food-borne pathogens Salmonella, Shigella, E. coli. using tandem mass spectrometry

254th National Meeting (2017)

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