Tandem Mass Spectrometry Analysis as an Approach to Delineate Genetically Related Taxa, with Specific Implication for Differentiating Escherichia coli from amongst the Complex Enterobacteriaceae Family

Aims: This work aims to evaluate the potential of GeLC-MS/MS to delineate taxa which are beyond the resolution of 16S rRNA and MALDI-TOF-MS identification, simultaneously discerning biomarkers of pathogenicity.

Methods and Results: 16S rRNA sequence analysis and MALDITOF- MS was performed to differentiate genetically closely related species from the family Enterobacteriaceae. In parallel GeLC-MS/ MS, using E. coli and related enteric bacteria as a model, was performed. Species specific peptides were identified and used to create an optimised identification database, against which a panel of test strains were analysed to determine the resolution of GeLCMS/ MS for bacterial identification and strain characterisation. The panel included amongst others pathogenic E. coli strains, of differing pathotype, including E. coli O104:H4. The test strains could be resolved to the species and pathotype (subspecies) in addition, specific features could be identified.

Conclusions: Using an optimised genome database and proteome profiling, we identified biomarkers that were specific for the test species and characterised strain-specific virulence factors. Significance and Impact of the

Study: This proof of concept study demonstrates that whole genome sequences and GeLC-MS/ MS have the potential to both identify and characterise pathogenic bacteria in a single assay, ushering in a new proteogenomic trend for microbial clinical diagnostics