Introduction
Shiga toxin-producing Escherichia coli (STEC) is a potentially deadly foodborne pathogen that causes diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) in humans worldwide (Smith, Fratamico, & Gunther, 2014). Enterohemorrhagic E. coli (EHEC) is a subgroup of STEC characterized by certain serogroups, which are often associated with outbreaks and severe illnesses. In the US alone, EHEC outbreaks have occurred nearly every year in the last 10 years (CDC, 2017; Fao/Who Stec Expert, 2019). The most common EHEC serogroups are O157, O26, O121, O103, O111, and O145. Although O157 is the predominant serogroup in STEC infections, recent reports have demonstrated that non-O157 STECs are emerging as more impactful pathogens associated with human infections and foodborne illness outbreaks. In several countries including the US, non-O157 associated human infection cases have exceeded that caused by O157 isolates (CDC, 2017; Valilis, Ramsey, Sidiq, & DuPont, 2018). Thus far, over 200 non-O157 serotypes of STEC have been discovered and linked to human diseases across the globe, among which the top six serogroups are: O26, O45, O103, O111, O121, and O145 (Conrad, Stanford, McAllister, Thomas, & Reuter, 2014). Certain serotypes that are usually associated with other pathotypes have recently caused outbreaks such as O104:H4 (Johura et al., 2016; Rasko et al., 2011).
STEC is defined by the production of one or more types of Shiga toxin, which, upon entry into host cells, inhibit the protein synthesis of host cells, eventually leading to cell death. Stx consists of two major types, namely, Stx1 and Stx2, which are closely related and encoded bystx 1 and stx 2, respectively (Melton-Celsa, 2014). Based on sequence and biological differences, stx 1 is classified into three subtypes (stx 1a, stx 1c, and stx 1d), and stx 2 into seven subtypes (stx 2a, stx 2b, stx 2c,stx 2d, stx 2e, stx 2f, and stx 2g) (Scheutz et al., 2012). Certain subtypes have been linked to human infections. For instance, STEC carryingstx 2a, stx 2c, or stx 2d is frequently associated with patients with HUS (Fruth, Prager, Tietze, Rabsch, & Flieger, 2015), whereas stx 2e is commonly detected in diseased pigs (Tseng et al., 2014). Aside from Shiga toxins, STEC strains utilize additional virulence factors that allow them to attach, colonize, invade, and cause damage. The locus of enterocyte effacement (LEE) pathogenicity island is responsible for producing attaching and effacing (A/E) lesions on intestinal epithelial cells. Specific contributing factors include intimin encoded by the eae gene, secreted effector proteins (Esp), an intimin receptor encoded by the tir gene, and others present in the LEE island (Galli, Miliwebsky, Irino, Leotta, & Rivas, 2010). However, LEE-negative STEC strains have been shown to be culprits in some human HUS cases. These could possess other genes responsible for attachment and colonization such as STEC autoagglutinating adhesin (saa ) (Paton, Srimanote, Woodrow, & Paton, 2001). Key virulence genes also consist of ehxA , espP ,etpD , toxB , katP , subA , saa , andsab genes. The EHEC hemolysin encoded by ehxA is a cytotoxin expressed during infection and is produced by a great majority of EHEC serogroups, mostly frequently linked to HUS (Bielaszewska, Aldick, Bauwens, & Karch, 2014; Schmidt, Kernbach, & Karch, 1996). The presence of ehxA along with stx 2 andeae represents a significant risk factor for severe clinical manifestations, particularly HUS (Boerlin et al., 1999).
As DNA sequencing cost decreases, whole-genome sequencing (WGS) has become increasingly popular in characterizing clinical/environmental bacterial isolates because the serotype, virulence genes, and antimicrobial resistance profile can be well predicted from whole-genome sequences. Additionally, DNA sequence data can provide much better resolution for strain discrimination than any subtyping method used so far for outbreak detection and bacteria tracking, rendering it the most powerful tool in revealing phylogenetic relationships (Bergholz, Moreno Switt, & Wiedmann, 2014; Dallman et al., 2015). Importantly, WGS-based analysis tools have been developed and applied to the characterization of STEC isolates (Ferdous et al., 2016; Gonzalez-Escalona & Kase, 2019; Gonzalez-Escalona et al., 2016). With sequencing coverages between 30 and 40, serotypes, virulence genes, and antibiotic resistance genes/sites can be accurately predicted, thereby providing a faster, cheaper, and better analytical method (Lindsey, Pouseele, Chen, Strockbine, & Carleton, 2016).
Cattle and sheep are ruminants that have been demonstrated to be important reservoirs for non-O157 STEC strains. Foods such as uncooked meat, unpasteurized milk, and vegetables contaminated by STEC strains have been frequently associated with human HC and HUS cases (CDC, 2017) and constitute a constant threat to human health (Djordjevic et al., 2004). Xinjiang Province is one of China’s largest provinces with vast pasture land and is thus known for breeding and husbandry of cattle and sheep. Large quantities of foods derived from cattle and sheep are consumed by locals as well as transported to neighboring regions. Cattle and/or sheep isolates from other parts of China were earlier studied using traditional molecular and typing methods (Bai et al., 2016; Fan et al., 2019). Previously, we isolated 56 STEC strains from cattle and sheep in Xinjiang, but the molecular characteristics and phylogeny of these STEC isolates remain untested. Thus, we here characterized this collection of STEC strains in detail based on whole-genome sequencing (WGS) and assessed their pathogenic potential.