Virulence Gene Profiles and Clonal Relationships of Escherichia coli O26:H11 Isolates from Feedlot Cattle as Determined by Whole-Genome Sequencing

Abstract

Escherichia coli O26 is the second most important enterohemorrhagic E. coli (EHEC) serogroup worldwide. Serogroup O26 strains are categorized mainly into two groups: enteropathogenic (EPEC) O26, carrying a locus of enterocyte effacement (LEE) and mostly causing mild diarrhea, and Shiga-toxigenic (STEC) O26, which carries the Shiga toxin (STX) gene (stx), responsible for more severe outcomes. stx-negative O26 strains can be further split into two groups. One O26 group differs significantly from O26 EHEC, while the other O26 EHEC-like group shows all the characteristics of EHEC O26 except production of STX. In order to determine the different populations of O26 E. coli present in U.S. cattle, we sequenced 42 O26:H11 strains isolated from feedlot cattle and compared them to 37 O26:H11 genomes available in GenBank. Phylogenetic analysis by whole-genome multilocus sequence typing (wgMLST) showed that O26:H11/H ؊ strains in U.S. cattle were highly diverse. Most strains were sequence type 29 (ST29). By wgMLST, two clear lineages could be distinguished among cattle strains. Lineage 1 consisted of O26:H11 EHEC-like strains (ST29) (4 strains) and O26:H11 EHEC strains (ST21) (2 strains), and lineage 2 (36 strains) consisted of O26: H11 EPEC strains (ST29). Overall, our analysis showed U.S. cattle carried pathogenic (ST21; stx 1 ؉ ehxA ؉ toxB ؉ ) and also potentially pathogenic (ST29; ehxA ؉ toxB ؉ ) O26:H11 E. coli strains. Furthermore, in silico analysis showed that 70% of the cattle strains carried at least one antimicrobial resistance gene. Our results showed that whole-genome sequence analysis is a robust and valid approach to identify and genetically characterize E. coli O26:H11, which is of importance for food safety and public health. IMPORTANCEEscherichia coli O26 is the second most important type of enterohemorrhagic E. coli (EHEC) worldwide. Serogroup O26 strains are categorized into two groups: enteropathogenic (EPEC) carrying LEE, causing mild diarrhea, and Shiga toxigenic (STEC) carrying the stx gene, responsible for more severe outcomes. However, there are currently problems in distinguishing one group from the other. Furthermore, several O26 stx-negative strains are consistently misidentified as either EHEC-like or EPEC. The use of whole-genome sequence (WGS) analysis of O26 strains from cattle in the United States (i) allowed identification of O26 strains present in U.S. cattle, (ii) determined O26 strain diversity, (iii) solved the misidentification problem, and (iv) screened for the presence of antimicrobial resistance and virulence genes in the strains. This study provided a framework showing how to easily and rapidly use WGS information to identify and genetically characterize E. coli O26:H11, which is important for food safety and public health.

Nanopore sequencing for fast determination of plasmids, phages, virulence markers, and antimicrobial resistance genes in Shiga toxin-producingEscherichia coli

“…Here, we tested the use of such a technique (i.e., Nanopore sequencing) to obtain the complete genomes of 3 unrelated STEC O26:H11 strains followed by subsequent comparisons to those obtained by PacBio and MiSeq WGS platforms. The 3 strains belonged to the EHEC O26 group in accordance with the O26:H11/- cgMLST scheme reported previously by Gonzalez-Escalona et al in 2016 (9).…”

“…The output assemblies from our MinION/CANU assembly were used for in silico detection of 95 described virulence genes for E. coli (39), which include genes reported from all pathotypes of pathogenic E. coli, with particular focus on EHECs. To test our hypothesis that MiSeq Nextera XT library sequencing was subpar for obtaining a complete representation of the STEC genomes (chromosome and plasmids), we compared results obtained using MinION assemblies against those obtained from MiSeq assemblies.…”

“…The phylogenetic relationships among E. coli O26:H11/H- strains were determined by a cgMLST analysis shown in Figure 2. This cgMLST was the same as previously published (9). The genome of E. coli strain 11368 (NC_013361.1) was used as a reference and has 4,554 genes.…”

“…Whole genome sequencing is an essential tool for characterizing and tracking pathogenic bacteria that may have contaminated the food supply as well as for identifying whether those bacteria carry virulence factors that could pose serious threats to public health (1,2). Closed bacterial genomes provide: 1) high-quality reference material that supports pathogen source tracking during a foodborne outbreak investigation, 2) important clues about the long-term evolution of enteric pathogens, 3) key insights into mechanisms and transmission of mobile elements conferring antimicrobial resistance, and 4) critical information about the contribution of DNA modifications on pathogenesis (2–9). These details are particularly important for understanding hemorrhagic pathogens such as E. coli O26:H11/- strains, which cause significant human morbidity and mortality worldwide (31,33,35,43).…”

“…These details are particularly important for understanding hemorrhagic pathogens such as E. coli O26:H11/- strains, which cause significant human morbidity and mortality worldwide (31,33,35,43). The genomes of O26:H11/- are very complex, containing many virulence genes, insertion sequences, phages, and plasmids; consequently, strains of the same lineage can possess significantly different content (7,9,35),(33,36–38). Missing the presence of some of these elements during an investigation can have large impacts on human health (e.g.…”