Shiga-toxin-producing Escherichia coli (STEC) and Enteropathogenic Escherichia coli (EPEC) are key diarrhoea-causing foodborne pathogens. We used proteomics to characterise the virulence and antimicrobial resistance protein profiles of three clinical pathogenic E. coli isolates (two EPEC [one was resistant to ciprofloxacin] and one STEC) cultured on CHROMagarTMSTEC solid media after minimal laboratory passage. We identified a total of 4767 unique peptides from 1630 proteins groups across all three clinical E. coli strains. Use of a label-free proteomic approach also allowed the identification of virulence and drug resistance proteins unique to each of the clinical isolates. The B subunit of Shiga toxin, ToxB, was uniquely detected in the STEC strain only and additionally several other virulence factors including SheA, OmpF, OmpC and OmpX were significantly more abundant in the STEC strain. The ciprofloxacin resistant EPEC isolate possessed reduced levels of key virulence proteins compared to the ciprofloxacin susceptible EPEC and STEC strains. We used PRM to validate the presence of biologically relevant proteins, which contribute to the unique phenotypic protein profile of each of the clinical E.coli isolates, across biologically-replicated cultures. Propagation of clinical isolates on a relevant solid medium followed by mass spectrometry analysis represents a convienient means to assess in a quantitative manner virulence factors and drug resistance determinants that might otherwise be lost through extensive in vitro passage in enteropathogenic bacteria.

We investigated the use of high-throughput mass spectrometry-based proteomics and transmission electron microscopy of isolates grown on CHROMagarTMSTEC solid media culture, to compare the proteomes and morphotypes of three clinical EPEC/STEC strains isolated from patients with diarrhoea. We selected 12 proteins of biological significance and examined their abundance using parallel reaction monitoring (PRM) in triplicate biological replicate cultures of each of the three clinical isolates. Based on our results, we propose a future workflow to differentiate the clinical strains using a targeted approach.

Single colonies of three E.coli clinical isolates cultured in triplicate on solid media.