A Targeted Sequencing Assay for Serotyping Escherichia coli Using AgriSeq Technology

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

doi:10.3389/fmicb.2020.627997

A Targeted Sequencing Assay for Serotyping Escherichia coli Using AgriSeq Technology

Front Microbiol. 2021 Jan 15:11:627997. doi: 10.3389/fmicb.2020.627997. eCollection 2020.

Affiliations

¹ U. S. Department of Agriculture, Eastern Regional Research Center, Agricultural Research Service, Wyndmoor, PA, United States.
² Thermo Fisher Scientific, Genetic Sciences Division, Austin, TX, United States.
³ Clear Labs, Menlo Park, CA, United States.
⁴ E. coli Reference Center, The Pennsylvania State University, University Park, PA, United States.

Abstract

The gold standard method for serotyping Escherichia coli 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 E. coli 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 (stx ₁, and stx ₂) and intimin (eae) were included. To validate the assay, genomic DNA was extracted from O-serogroup and H-type standard strains and from Shiga toxin-producing E. coli, 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 via the SeroType Caller™ software for identification of O-serogroup, H-type, and presence of stx ₁ , stx ₂, and eae. We successfully identified 169 O-serogroups and 41 H-types. The assay also routinely detected the presence of stx _1a,c,d (3 of 3 strains), stx _2c-e,g (8 of 8 strains), stx _2f (1 strain), and eae (6 of 6 strains). Taken together, the high-throughput, sequence-based method presented here is a reliable alternative to antisera-based serotyping methods for E. coli.

Keywords: AgriSeq technology; E coli; O-antigen; molecular serotyping; targeted sequencing.