Description:
<jats:p>The gold standard method for serotyping<jats:italic>Escherichia coli</jats:italic>has relied on antisera-based typing of the O- and H-antigens, which is labor intensive and often unreliable. In the post-genomic era, sequence-based assays are potentially faster to provide results, could combine O-serogrouping and H-typing in a single test, and could simultaneously screen for the presence of other genetic markers of interest such as virulence factors. Whole genome sequencing is one approach; however, this method has limited multiplexing capabilities, and only a small fraction of the sequence is informative for subtyping or identifying virulence potential. A targeted, sequence-based assay and accompanying software for data analysis would be a great improvement over the currently available methods for serotyping. The purpose of this study was to develop a high-throughput, molecular method for serotyping<jats:italic>E. coli</jats:italic>by sequencing the genes that are required for production of O- and H-antigens, as well as to develop software for data analysis and serotype identification. To expand the utility of the assay, targets for the virulence factors, Shiga toxins (<jats:italic>stx</jats:italic><jats:sub>1</jats:sub>, and<jats:italic>stx</jats:italic><jats:sub>2</jats:sub>) and intimin (<jats:italic>eae</jats:italic>) were included. To validate the assay, genomic DNA was extracted from O-serogroup and H-type standard strains and from Shiga toxin-producing<jats:italic>E. coli</jats:italic>, the targeted regions were amplified, and then sequencing libraries were prepared from the amplified products followed by sequencing of the libraries on the Ion S5™ sequencer. The resulting sequence files were analyzed<jats:italic>via</jats:italic>the SeroType Caller™ software for identification of O-serogroup, H-type, and presence of<jats:italic>stx</jats:italic><jats:sub>1</jats:sub><jats:italic>, stx</jats:italic><jats:sub>2</jats:sub>, and<jats:italic>eae</jats:italic>. We successfully identified 169 O-serogroups and 41 H-types. The assay also routinely detected the presence of<jats:italic>stx</jats:italic><jats:sub>1a,c,d</jats:sub>(3 of 3 strains),<jats:italic>stx</jats:italic><jats:sub>2c−e,g</jats:sub>(8 of 8 strains),<jats:italic>stx</jats:italic><jats:sub>2f</jats:sub>(1 strain), and<jats:italic>eae</jats:italic>(6 of 6 strains). Taken together, the high-throughput, sequence-based method presented here is a reliable alternative to antisera-based serotyping methods for<jats:italic>E. coli</jats:italic>.</jats:p>