AUTHOR=Elder Jacob R. , Fratamico Pina M. , Liu Yanhong , Needleman David S. , Bagi Lori , Tebbs Robert , Allred Adam , Siddavatam Prasad , Suren Haktan , Gujjula Krishna Reddy , DebRoy Chitrita , Dudley Edward G. , Yan Xianghe 

TITLE=A Targeted Sequencing Assay for Serotyping Escherichia coli Using AgriSeq Technology

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.627997

DOI=10.3389/fmicb.2020.627997

ISSN=1664-302X

ABSTRACT=<p>The gold standard method for serotyping <italic>Escherichia coli</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 <italic>E. coli</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 (<italic>stx</italic><sub>1</sub>, and <italic>stx</italic><sub>2</sub>) and intimin (<italic>eae</italic>) were included. To validate the assay, genomic DNA was extracted from O-serogroup and H-type standard strains and from Shiga toxin-producing <italic>E. coli</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 <italic>via</italic> the SeroType Caller™ software for identification of O-serogroup, H-type, and presence of <italic>stx</italic><sub>1</sub><italic>, stx</italic><sub>2</sub>, and <italic>eae</italic>. We successfully identified 169 O-serogroups and 41 H-types. The assay also routinely detected the presence of <italic>stx</italic><sub>1a,c,d</sub> (3 of 3 strains), <italic>stx</italic><sub>2c−e,g</sub> (8 of 8 strains), <italic>stx</italic><sub>2f</sub> (1 strain), and <italic>eae</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 <italic>E. coli</italic>.</p>