A targeted sequencing assay for serotyping Escherichia coli using AgriSeq technology

Authors: ELDERS, JACOB - Oak Ridge Institute For Science And Education (ORISE); FRATAMICO, PINA - Collaborator; Liu, Yanhong; NEEDLEMAN, DAVID - Collaborator; Bagi, Lori; TEBBS, ROBERT - Thermo Fisher Scientific; ALLRED, ADAM - Thermo Fisher Scientific; SIDDAVATAM, PRASAD - Clear Labs; SUREN, HAKTAN - Clear Labs; GUJJULA, KRISHNA - Clear Labs; DEBROY, GUJJULA - Pennsylvania State University; DUDLEY, EDWARD - Pennsylvania State University; Yan, Xianghe

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/21/2020
Publication Date: 1/15/2021
Citation: Elders, J., Fratamico, P., Liu, Y., Needleman, D., Bagi, L.K., Tebbs, R., Allred, A., Siddavatam, P., Suren, H., Gujjula, K., Debroy, G., Dudley, E.G., Yan, X. 2021. A targeted sequencing assay for serotyping Escherichia coli using AgriSeq technology. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2020.627997.
DOI: https://doi.org/10.3389/fmicb.2020.627997

Interpretive Summary: The bacterium, Escherichia coli, causes a variety of diseases in humans and animals, and many non-harmful E. coli types (serogroups) also exist. Traditionally, a procedure called serotyping has been used to distinguish among the different E. coli serogroups. This procedure, which relies on the use of antibodies raised in rabbits against different surface polysaccharides of E. coli can only be performed in specialized laboratories, is labor intensive and may require several days to complete, and one antiserum can react with multiple E. coli serogroups, rendering identification of the specific E. coli type difficult. Thus, due to the lack of simple, rapid, and reliable methods for detection and identification of harmful and non-harmful E. coli types, the incidence of disease caused by harmful strains of E. coli may be underestimated, and epidemiological studies are difficult to perform. The aim of our work was to develop a genetic-based method known as AgriSeq that is based on targeting specific sequences of genes that are involved in synthesis of E. coli surface structures (O-antigen and flagellar H-antigen). Following amplification and sequencing of the specific regions, the developed software is used to identify the specific E. coli type. Hundreds of samples can be tested simultaneously making the method very cost-effective. Furthermore, the assay also identifies the presence of several genes involved in causing illness by a specific type of pathogenic E. coli. The method correctly identified 169 O-serogroups and 41 H-types. Taken together, this high-throughput, sequence-based method is a rapid, accurate, and reliable alternative to antisera-based serotyping methods for E. coli.

Technical 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 (stx1, and stx2) 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 stx1, stx2, and eae. We successfully identified 169 O-serogroups and 41 H-types. The assay also routinely detected the presence of stx1a, c, d (3 of 3 strains), stx2c-e, g (8 of 8 strains), stx2f (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.