ABSTRACT

Shiga toxin–producing Escherichia coli (STEC) O157:H7/nonmotile and some non-O157 STEC strains are foodborne pathogens. In response to pork-associated O157 STEC outbreaks in Canada, we investigated the occurrence of STEC in Canadian retail raw ground pork during the period of 1 November 2014 to 31 March 2016. Isolated STEC strains were characterized to determine the Shiga toxin gene (stx) subtype and the presence of virulence genes encoding intimin (eae) and enterohemorrhagic E. coli hemolysin (hlyA). O157 STEC and non-O157 STEC strains were isolated from 1 (0.11%) of 879 and 13 (2.24%) of 580 pork samples, respectively. STEC virulence gene profiles containing both eae and hlyA were found only in the O157 STEC (stx2a, eae, hlyA) isolate. The eae gene was absent from all non-O157 STEC isolates. Of the 13 non-O157 STEC isolates, two virulence genes of stx1a and hlyA were found in four (30.8%) O91:H14 STEC isolates, whereas one virulence gene of stx2e, stx1a, and stx2a was identified in five (38.5%), two (15.4%), and one (7.7%) STEC isolates, respectively, of various serotypes. The remaining non-O157 STEC isolate carried stx2, but the subtype is unknown because this isolate could not be recovered for sequencing. O91:H14 STEC (stx1a, hlyA) was previously reported in association with diarrheal illnesses, whereas the other non-O157 STEC isolates identified in this study are not known to be associated with severe human illnesses. Virulence gene profiles identified in this study indicate that the occurrence of non-O157 STEC capable of causing severe human illness is rare in Canadian retail pork. However, O157 STEC in ground pork can occasionally occur; therefore, education regarding the potential risks associated with STEC contamination of pork would be beneficial for the public and those in the food industry to help reduce foodborne illnesses.

HIGHLIGHTS
  • O157 STEC was isolated from 1 (0.11%) of 879 ground pork samples.

  • Non-O157 STEC was isolated from 13 (2.24%) of 580 ground pork samples.

  • O157 STEC was the only STEC isolate carrying virulence genes stx2a, eae, and hlyA.

  • Non-O157 STEC isolates carried single stx genes stx1a, stx2e, or stx2a.

  • The presence of virulent STEC in Canadian pork is uncommon.

Shiga toxin–producing Escherichia coli (STEC) is a diverse group of E. coli capable of producing Shiga toxins, comprising more than 200 E. coli serotypes that are often described as E. coli O157:H7/nonmotile (O157 STEC) and non-O157 STEC (serotypes other than E. coli O157:H7/nonmotile). O157 STEC is an important foodborne pathogen that causes numerous outbreaks of human illness ranging in severity from uncomplicated diarrhea to severe outcomes of hemorrhagic colitis and hemolytic uremic syndrome (HUS) (54). Several serotypes of non-O157 STEC also cause outbreaks and sporadic cases of foodborne illness similar in severity to those caused by O157 STEC and have emerged as important foodborne pathogens worldwide (40, 51, 56). More often referred to by their O serogroups, these have been identified as “priority” or “top six” non-O157 STEC and can vary somewhat among regions or countries. In Canada the six priority non-O157 STEC serogroups are O26, O103, O111, O117, O121, and O145 (21), and in the United States they are O26, O45, O103, O111, O121, and O145 (40).

The defining feature and a major virulence factor of all STEC strains is the production of Shiga toxin 1 (Stx1) and/or Stx2, which are further categorized phylogenetically into three subtypes of Stx1 (1a, 1c, and 1d) and seven subtypes of Stx2 (2a, 2b, 2c, 2d, 2e, 2f, and 2g) (63, 71). Severe human illnesses such as hemorrhagic colitis and HUS are more often associated with STEC that produce Stx2 (28, 36), especially three Stx2 subtypes, Stx2a, Stx2c, and Stx2d (34, 36, 55). Other Stx2 subtypes, Stx2e, Stx2f, and Stx2g, are more frequent in animal and environmental STEC than human STEC, with Stx2e being the major virulence factor of E. coli associated with edema disease in swine (41). Another important factor in many virulent human STEC strains is the secreted adhesive protein intimin that is encoded by the eae gene and that contributes to the intimate adherence of STEC to enterocytes and formation of the attaching and effacing intestinal lesions typical of STEC and enteropathogenic E. coli (54). STEC that possesses both the eae and stx2a genes is more strongly associated with the hemorrhagic colitis and HUS of severe STEC infections (28, 34). Some STEC strains lacking the eae gene can also cause severe infections through other mechanisms of intestinal attachment (11, 15). Among other virulence factors of O157 STEC and some non-O157 STEC is the plasmid-borne E. coli enterohemolysin, or enterohemorrhagic E. coli (EHEC) hemolysin (EHEC-Hly) (10). It is encoded by the EHEC-hlyA (hlyA) gene and may contribute to the virulence of these STEC strains' hemolytic activities. Over 86% of STEC illnesses, including HUS, in North America are caused by STEC strains that possess both the eae and hlyA genes (18).

Human STEC infections are often caused by ingestion of STEC-contaminated foods of animal origin. Ruminants, in particular cattle, are regarded as a principal natural reservoir of STEC (37, 41), with foods such as undercooked ground beef and raw milk being the major vehicles of foodborne STEC outbreaks. Other ruminants, such as sheep, goats, and deer, may also act as reservoirs of STEC (61, 67). Nonruminant animals, like swine, turkeys, wild birds, and insects, can also harbor STEC and are capable of transmitting STEC to other animals and humans (33, 75). However, STEC outbreaks linked to foods from nonruminant animals, including pork, are relatively uncommon. Since 2004 there have been several documented O157 STEC outbreaks involving pork (77), including in Italy in 2004 (23), in Ontario, Canada, in 2011 (74), and in Alberta, Canada, in 2014 (48), 2016 (1), and 2018 (2). There have been sporadic reports of non-O157 STEC–associated outbreaks and illnesses involving pork: a single non-O157 STEC (O111, unknown stx genes) outbreak related with barbecue pork (60) and one non-O157 STEC (O8:H19, stx2e) illness case related with pork (70).

The reported prevalence of O157 and non-O157 STEC by isolation in pork in several countries varies from 0 to 2.5% (7, 17, 25, 30, 49, 52, 53, 69) and 0 to 5.2% (4, 7, 30, 52, 53), respectively. However, recent data regarding the prevalence and characteristics of STEC in Canadian retail pork are limited. Given this information gap and in response to the 2014 and 2011 O157 STEC outbreaks associated with pork in Alberta and Ontario, Canada, the Canadian Food Inspection Agency (CFIA) conducted a targeted survey of STEC in raw ground pork. The objectives of this study were to gain current baseline information on the occurrence of O157 and non-O157 STEC in Canadian retail raw ground pork, and to characterize the STEC isolates with respect to their complement of stx, eae, and hlyA genes and their human virulence potential.

Sample collection

A total of 879 samples of prepackaged raw ground pork weighing at least 250 g were collected on a weekly basis between 1 November 2014 and 31 March 2015 (group 1; 299 samples) and between 1 April 2015 and 31 March 2016 (group 2; 580 samples). Group 1 samples were tested only for O157 STEC as an immediate response to the O157 STEC outbreak associated with pork in Alberta, Canada, in 2014 (48). Group 2 samples were tested for both O157 STEC and non-O157 STEC as a planned 1-year survey. All samples were collected from 11 major cities located across Canada. The percentage of samples collected from each city was roughly proportional to the population of the province in which they were located in relation to the total population of Canada. The cities from which samples were collected, and the percentage collected from each, were as follows: Halifax (6.3%), St. John (2.6%), Montreal (15.5%), Quebec City (6.4%), Toronto (22.1%), Ottawa-Gatineau (12.3%), Vancouver (12.7%), Kelowna (1.3%), Calgary (11.0%), Saskatoon (3.3%), and Winnipeg (6.6%). All samples were promptly transported in insulated coolers with ice packs to CFIA laboratories for testing within 48 h of collection.

O157 STEC enrichment, identification, isolation, and confirmation

Samples from both group 1 (299 samples) and group 2 (580 samples) were tested for O157 STEC. O157 STEC enrichment, detection, isolation, and confirmation were conducted using methods published in Health Canada's The Compendium of Analytical Methods (47) (Supplemental Appendix I). Briefly, each sample (65 g of ground pork) was enriched in nine volumes of a modified tryptic soy broth with 20 mg/L novobiocin (mTSB-n; Oxoid CM0989, Oxoid Ltd., Basingstoke, Hampshire, England) at 42°C for 18 to 24 h (for Microbiological Analysis of Food Laboratory Procedures [MFLP]-30) (42, 47) or nine volumes of BAX system MP media (Hygiena, Mississauga, Ontario, Canada) at 42°C for 9 to 24 h (for method MFLP-76) (46, 47). Following enrichment, samples were first screened for O157 STEC using either MFLP-30 (42), a rapid PCR screening method targeting O157 STEC–specific markers (kit 2004, Hygiena) or MFLP-76 (46), a rapid real-time PCR screening method targeting O157 STEC–specific markers (kit 2000, Hygiena). Putative positive enrichments were processed for isolation by immunomagnetic separation (Thermo Fisher Scientific, Waltham, MA), followed by selective plating and isolation as described in the Microbiological Analysis of Food Health Protection Branch methods [MFHPB]-10 (44, 47). Putative O157 isolates were confirmed using traditional biochemical testing as described in MFHPB-10 (44, 47) or by a rapid colony hybridization method, MFLP-22 (43, 47). This rapid confirmation method identifies definitive gene sequences associated with this pathogen, targeting O157:H7 and stx1 and stx2. Confirmed O157 STEC isolates were further characterized by whole genome sequencing (WGS) as described in “Genomic characterization of STEC isolates.”

Non-O157 STEC enrichment, identification, isolation, and confirmation

Group 2 (580) samples were tested for non-O157 STEC. Non-O157 STEC testing was performed using method MFLP-52 (45). Certain samples were further characterized using an expanded multiplex PCR and cloth-based hybridization array system (PCR-CHAS) that targets STEC serogroups O157, O26, O45, O103, O111, O121, and O145 (13) (Appendix I). Briefly, each sample (65 g of ground pork) was enriched in a modified TSB (Oxoid CM0989) at the sample-to-buffer ratio of 1:9 with delayed addition of the antimicrobials of vancomycin (10 μg/mL) and cefsulodin (3 μg/mL) (39) as described in MFLP-52 (45, 47). Following enrichment, the broth was screened for the genes encoding stx1 and stx2 by PCR. stx-positive enrichment broth cultures were subsequently plated onto Rainbow agar O157 (Biolog 80102, Biolog Inc., Hayward, CA), and colonies were screened batch-wise by the stx-PCR to identify stx-positive isolates. The isolates were subsequently confirmed by a multiplex PCR-CHAS, which simultaneously identifies the presence of genes encoding virulence factors Stx1 (stx1), Stx2 (stx2), intimin (eae), and EHEC-hemolysin (hlyA), as well as the O serogroup markers O157, O26, O103, O111, and O145 (14, 45), or by an expanded multiplex PCR-CHAS, which simultaneously identifies the presence of genes stx1, stx2, and eae, as well as the O serogroup markers O157, O26, O45, O103, O111, O121, and O145 (13). STEC isolates with or without the O serogroups identified were all further characterized by WGS, as described in “Genomic characterization of STEC isolates.”

Genomic characterization of STEC isolates

STEC isolates obtained from STEC (O157 and non-O157)–positive samples were subjected to characterization by WGS to determine serotype and assess the presence of the selected virulence genes (stx subtypes, eae, hlyA) (20, 58). Briefly, bacterial isolates were cultured in brain heart infusion broth (Oxoid) for 3 to 6 h at 37°C. Genomic DNA was extracted using the Maxwell 16 cell SEV DNA purification kit (Promega, Madison, WI) and quantified using the Quant-it High-Sensitivity DNA assay kit (Life Technologies Inc., Burlington, Ontario, Canada). Sequencing libraries were constructed from 1 ng of gDNA using the Nextera XT DNA sample preparation kit and the Nextera XT Index kit (Illumina, Inc., San Diego, CA) and sequenced on the MiSeq using a v3 reagent kit (Illumina, Inc.). Genomic sequencing was performed on the Illumina MiSeq Platform (Illumina, Inc.) with a 600-cycle MiSeq reagent kit v3 (Illumina, Inc.). Sequencing errors in reads were corrected using Quake version 0.3 with a k-mer size of 15 (57). Reads were assembled de novo using SPAdes v. 3.9.1 (5), and contigs shorter than 1,000 bp were removed from the assemblies. Presence of virulence genes within the assembled genomes was determined based on detection of e-probe sequences for genes encoding Shiga toxin 1 (stx1) and 2 (stx2), intimin (eae), EHEC-hemolysin (hlyA), and O-serogroup markers, as previously described (13, 14, 58). Shiga toxin subtypes were detected from raw reads using the V-typer tool as previously described (20). Detection of a comprehensive set of full-length virulence genes in assembled genomes was performed using the VirulenceFinder tool provided by the Center for Genomic Epidemiology (CGE; https://cge.cbs.dtu.dk/services/VirulenceFinder/) using default parameters (90% identity, 60% minimum length) (50). Only results for stx, eae, and hlyA are reported here. Similarly, serotypes were identified using the CGE tool Serotype Finder version 1.1 (https://cge.cbs.dtu.dk/services/SerotypeFinder/) and default parameters (85% identity, 60% minimum length) (50). Raw data and assemblies have been deposited at DDBJ/EMBL/GenBank under BioProject PRJNA454819. Accession numbers are listed in Supplemental Table 1.

Prevalence of O157 and non-O157 STEC in raw ground pork

All 879 ground pork samples (groups 1 and 2) were analyzed for O157 STEC, and 580 ground pork samples (group 2) were analyzed for non-O157 STEC (Table 1). O157 STEC was isolated from one of the 879 pork samples, resulting in an estimated prevalence of 0.11% with a 95% confidence interval (CI) of 0.02 to 0.64% (Table 1). Non-O157 STEC was isolated from 13 of the 580 pork samples, resulting in an estimated prevalence of 2.24% (95% CI [1.31, 3.80]) (Table 1).

TABLE 1

Frequency of O157 and non-O157 STEC isolation from raw ground pork samples

Frequency of O157 and non-O157 STEC isolation from raw ground pork samples
Frequency of O157 and non-O157 STEC isolation from raw ground pork samples

Characterization of STEC isolates derived from raw ground pork

One O157 STEC isolate and 13 non-O157 STEC isolates were identified among 14 STEC-positive pork samples (Table 2). Twelve STEC isolates consisted of eight unique serotypes, and the two remaining STEC isolates were untypeable for O serogroup antigens (Ount). The stx2 gene subtype for one STEC isolate (O45) is unknown because it could not be recovered for further characterization by WGS. Three stx gene subtypes, stx2a, stx1a, and stx2e, were identified among the other 13 STEC isolates (Table 2). The stx2a subtype was detected in one O157:H7 STEC and one O132:H18 STEC isolate, whereas the stx1a subtype was identified in four O91:H14 STEC and two O163:H19 STEC isolates. Significantly, the stx2e subtype was identified in five different non-O157 STEC serotypes. With the exception of serotypes O157:H7 (stx2a, eae, hlyA) and O91:H14 (stx1a, hlyA), the virulence genes eae and hlyA were not identified in any other STEC isolates from ground pork (Table 2).

TABLE 2

Characterization of STEC isolates derived from pork and their respective risk levels

Characterization of STEC isolates derived from pork and their respective risk levels
Characterization of STEC isolates derived from pork and their respective risk levels

Based on the virulence gene profile and in association with clinical illness severity, the Joint Food and Agriculture Organization of the United Nations, World Health Organization (FAO/WHO) Expert Meeting on Microbiological Risk Assessment has categorized the potential risk to human health associated with STEC strains in food into five levels, from level 1 (highest) to level 5 (lowest) (FAO/WHO STEC risk categories) (34). O157 STEC is a known pathogen that can cause human illness ranging in severity from uncomplicated diarrhea to bloody diarrhea to severe outcomes of hemolytic uremic syndrome (HUS) (34) (Table 2). Given that the O157 STEC isolate derived from raw ground pork in our study carried the stx2a and eae virulence genes, the O157 STEC (stx2a, eae, hlyA) isolate was classified as belonging to the highest risk level (level 1) according to the FAO/WHO STEC risk categories (Table 2). Of the non-O157 STECs identified in this study, only O91:H14 STEC carrying the stx1a and hlyA virulence gene profile was previously reported to be associated with diarrheal illness (8, 29), and provided that this isolate did not carry eae, the O91:H14 STEC (stx1a, hlyA) isolate was considered to belong to the lowest risk level (level 5) as per the FAO/WHO STEC risk categories (Table 2). Other non-O157 STECs identified in this study were not found to be reported in association with human illness at the time of publication, and because they carried a single stx2a, or stx1a, or stx2e gene, they belonged to the lowest risk level (level 5) as per the FAO/WHO STEC risk categories (Table 2). The stx2 gene subtype of the O45 STEC isolate is unknown, and, therefore, it could not be determined which of the FAO/WHO STEC risk categories it belonged to.

The 14 STEC-positive pork samples were processed in a variety of establishments located across Canada and were sampled from various retail locations over the course of the study (see Table 3). For example, the four O91:H14 isolates were derived from four ground pork samples processed at two different establishments located in Quebec and British Columbia. Two samples, one conventional and one organic, from the establishment located in Quebec were collected from the same retail store in Lachine, Quebec, in July and September 2015, respectively. The other two samples from the establishment located in British Columbia were collected from retail stores located in Vancouver, British Columbia, and Calgary, Alberta, in November 2015 and January 2016, respectively. The two O163:H19 isolates were derived from two ground pork samples processed at two different establishments located in Quebec and Ontario, and the two samples were collected from different retail stores located in Mississauga, Ontario, in October 2015 and February 2016, respectively. The O157 STEC isolate was derived from a ground pork sample that was processed at a meat market located in Vancouver, British Columbia. The CFIA conducted appropriate follow-up actions, including a food safety investigation and product recalls related to the O157 STEC–positive finding (19); however, the source of O157 STEC could not be determined.

TABLE 3

Establishment and sample collection locations of STEC-positive samples

Establishment and sample collection locations of STEC-positive samples
Establishment and sample collection locations of STEC-positive samples

O157 STEC in ground pork

The O157 STEC isolate derived from ground pork in this study carried a virulence gene profile of stx2a, eae, hlyA, which can cause severe human illnesses. The estimated prevalence of O157 STEC in raw ground pork in this study was 1 (0.11%) of 879 with a 95% CI of 0.02 to 0.64%, which falls in the lower range of previously reported worldwide estimated prevalence of 0 to 2.5% by isolation (7, 17, 25, 30, 49, 52, 53, 69). In earlier studies, O157 STEC was not isolated from ground pork taken from abattoirs located in Ontario, Canada (235 samples) (69), whereas it was found in 4 (1.5%) of 264 retail ground pork samples including 1 of 14 ground pork samples originating from Calgary, Canada, in a U.S. study conducted in the same time period in the mid-1980s (25). More recently, O157 STEC was isolated from 2 (2.5%) of 80 retail pork samples in South Korea in 2000 to 2002 (49). O157 STEC was not isolated from pork samples (0 of 498) collected from pork cutting plants in France (17), nor from retail ground pork in Switzerland (0 of 189) (30), Italy (0 of 465) (7), and the United States (0 of 231 (52) and 0 of 396 (53)). The prevalence of O157 STEC in retail ground pork (0.11%, 95% CI [0.02 to 0.64%]) from this study is similar to that observed in Canadian retail ground beef (0.1%) tested in 2012 and 2014 (68, 79), and it appears to be lower than ground beef from Canadian processing plants during 2008 to 2011 (lowest positive rate was 0.24%) (68) and U.S. processing plants during 2007 to 2016 (517 of 202,890, 0.25%) (79).

Swine, unlike cattle, are not considered a prominent natural reservoir for STEC virulent to humans, but close-to-market swine may carry O157 STEC when frequently exposed to O157 STEC from environmental sources, such as cattle feedlots (67). Early studies pointed out that a high rate of O157 STEC was found in ground meat (beef and pork) originating from Calgary, Alberta, Canada (25). Following three O157 STEC outbreaks in Alberta, Canada, that were associated with ground pork, a focused study was conducted in which O157 STEC was isolated from 7 (1.4%) of 504 swine colon fecal samples from healthy market swine of Albertan farms (27). Other studies have also detected O157 STEC in swine colon fecal samples obtained at slaughter facilities in the United States (32). O157 STEC was recovered from swine manure samples in a longitudinal cohort study in the United States (22). Additionally, experimentally infected swine have been shown to harbor and shed O157 STEC for up to 2 months postinfection (16). These studies suggest that swine are capable of acting as a host or a temporary host for O157 STEC and, consequently, can play a role in O157 STEC transmission (67). Note that the O157 STEC isolate obtained from this study does not match any historical isolates recovered from the CFIA (2009 to the present), nor did it cluster with any of the isolates currently published in the NCBI Pathogen Detection database (https://www.ncbi.nlm.nih.gov/pathogens/; accessed 8 June 2021) (66).

Non-O157 STEC in ground pork

The occurrence of non-O157 STEC in ground pork in this study was 2.24% (95% CI [1.30, 3.80]). The prevalence of non-O157 STEC in pork has been reported in other studies and varies from 0 to 5.2% by isolation (4, 7, 30, 52, 53). Currently, there is no standard laboratory method for all non-O157 STEC serotypes due to their broad genetic and biochemical diversity. The reported prevalence of non-O157 STEC in pork may be underestimated due to the difficulties with isolation from food samples. As observed in an earlier study of STEC in ground pork carried out by Read et al. (69), 10.6% of 235 samples were stx positive; however, only 3.8% of the non-O157 STEC isolates were recovered from the pork samples. In another recent study, authors also pointed out that it was difficult to isolate STEC from food samples, and in their study neither non-O157 STEC nor O157 STEC were isolated from 19 (2.8%) of 675 stx-positive pork samples (26). The CFIA testing method has been optimized for the detection of selected priority non-O157 STEC (i.e., O26, O45, O103, O111, O121, and O145) over the detection of other nonpriority STEC (13, 14, 38, 39, 45), although other O serogroups can be identified through WGS.

Swine, while not considered a prominent natural reservoir for STEC virulent in humans, are an important natural reservoir of non-O157 STEC. Non-O157 STEC isolates in swine colon fecal samples obtained at slaughter facilities (26) and in swine manure samples (3, 22, 64, 75) have been reported. A longitudinal study was conducted on U.S. farms to investigate fecal shedding of STEC of three cohorts of finishing swine (n = 50 per cohort) from 10 weeks of age until 24 weeks of age. The prevalence of non-O157 STEC in manure samples was highest (between 39.5 and 59.2%) in swine ranging from 14 to 18 weeks of age, and the duration of non-O157 STEC shedding ranged from 14 to 56 days (76). Most (85 to 100%) non-O157 STEC isolates derived from swine manure carried the stx2e gene (3, 6, 64, 75, 76), and other stx gene subtypes such as stx1a, in combination with or without other virulence genes, were also reported (6, 22, 75). These studies suggest that swine are capable of acting as a host for such non-O157 STEC and, consequently, can play a role in non-O157 STEC transmission (67).

Non-O157 STEC possessing stx1a or stx2a isolated from ground pork

Of the 13 non-O157 STEC isolates from ground pork, stx1a was the most frequently (6 of 13, 46.2%) identified stx gene and was found in four O91:H14 (stx1a, hlyA) and two O163:H19 STECs (stx1a) isolates, respectively. O91:H14 carrying virulence genes of stx1a and hlyA was isolated in this study, from retail ground pork in the Washington, DC, area (52), in retail pork from Korea (59), and from the manure of clinically healthy swine in the United States (6, 35). O91:H14 STEC (stx1a, hlyA) was one of the non-O157 STECs frequently reported from patients with diarrhea (8, 29, 59, 62) or healthy human carriers (3, 6). One recent study found that O91:H14 STEC isolates derived from beef and pork were all categorized as belonging to the STEC lineage ST33 (sequence type 33); these include O91:H14 STEC isolates from patients with mild diarrhea or from asymptomatic carriers (59). Although belonging to the ST33 lineage, the O91:H14 STEC isolates derived from beef (stx1a, stx2b, hlyA) carried two stx subtypes and genes of cytotoxins and adhesins, such as subAB (subtilase cytosine), saa (STEC auto-agglutinating adhesin), and iha (iron-regulated adhesin), whereas the O91:H14 STEC (stx1a, hlyA) derived from pork was found to carry a single stx subtype and did not carry the genes subAB, saa, and iha (59). The four O91:H14 (stx1a, hlyA) isolates obtained in this pork study did not cluster with any of the isolates currently published in the NCBI Pathogen Detection database (66). The O91:H14 (stx1a, hlyA) isolates from this pork study may be associated with sporadic human diarrheal illness; they belong to the lowest risk level (level 5) as per the FAO/WHO STEC risk categories (34) because the eae virulence gene was lacking.

O163:H19 STEC carrying stx1a was found in ground pork in this study and in healthy swine manure in U.S. studies (6, 22). Human infections due to O163:H19 serotype were rarely reported (8, 40, 80). In one study, it was not specifically reported whether the serotype associated with a clinical diarrhea case carried the stx1a gene alone or in addition to the virulence gene hlyA (8). The serotype O132:H18 (stx2a) isolated from pork in our study was the only serotype other than E. coli O157:H7 that carried the stx2a gene. Serotype O132:H18 (not possessing stx or eae) was reported as an E. coli nonpathogenic to humans (24). Both O163:H19 (stx1a) and O132:H18 (stx2a) isolated from raw ground pork in this study were classified as belonging to the lowest risk level (level 5) according to the FAO/WHO STEC risk categories (34) because neither isolate possessed the eae virulence gene.

Non-O157 STEC possessing stx2e and an unknown stx2 subtype isolated from ground pork

Of the 13 non-O157 STEC isolates from ground pork, stx2e was the second most frequently (5 of 13, 38.5%) identified stx gene in five various serotypes. The STEC serogroups O121 (H10, stx2e) and O45 (stx2) found in raw ground pork in the current study are the same serogroups of known priority non-O157 STEC in Canada (O121) and in the United States (O121 and O45). However, their virulence gene profiles differ from those associated with human illness. The O121:H19 serotype associated with human illnesses possesses both the stx2a and eae virulence genes (stx2a, eae) (8, 29). The O45:H2 serotype associated with human illnesses (29, 40) mostly possesses the stx1 gene and both the eae and hlyA genes (O45:H2, stx1, eae, hlyA) (18). The serotype O86:H32 (stx2e) isolated from ground pork in this study and from manure of healthy pigs (22) is not known to be associated with human illnesses. The O-type could not be identified for two of the STEC stx2e strains recovered from this study (Ount:H26 and Ount:H38, Table 2). This is likely due to the absence of matching serotypes in the database upon which the WGS-based serotyping tool relies (50). The STEC isolates possessing the stx2e gene isolated from raw ground pork in this study have not been reported in association with human illnesses (29, 78) (Table 2). Unlike STEC carrying stx2a (in combination with other virulence genes such as eae) that have been associated with human STEC infections and severe HUS (18, 29, 34), STEC (stx2e) isolates derived from swine origin often lack eae and hlyA virulence genes. Critical virulence genes in E. coli are commonly associated with mobile genetic elements (12). For example, the eae gene is part of the Locus of Enterocyte Effacement pathogenicity island that encodes proteins that play an important role in intestinal colonization. The EHEC-hlyA gene is typically plasmid encoded. The Shiga toxins are encoded by lambdoid bacteriophages integrated into the bacterial chromosome, where they remain stably present as prophages. Under bacterial stress, phage particles may be produced and released to infect new E. coli hosts. The STEC (stx2e) strains were not found to have the inducible stx-carrying phages, suggesting they may have different ecological origins from other stx-carrying E. coli strains (65). stx2e was also found to have an altered receptor specificity that differs from Shiga toxins (9). Thus STEC strains carrying stx2e have only occasionally been found in human STEC isolates (8, 72, 73) from asymptomatic carriers (8, 73). STEC isolates carrying stx2e in combination with other virulence genes have been sparsely reported, such as in patients with mild diarrhea (O91:H21, stx1a+stx2e) (8, 73) or acute diarrhea (O8:H19, stx2e) (70) and in one transplant patient with HUS (O51:H49, stx2e+eae) (31). Currently available information suggests that the virulence potential of identified non-O157 STEC isolates possessing the stx2e gene and lacking other virulence genes such as eae in this study is low and unlikely to cause severe human illness. Therefore the non-O157 STEC strains possessing stx2e isolated from pork in our study have been identified as belonging to the lowest level of risk (level 5) as per the FAO/WHO STEC risk categories, with a potential to cause diarrhea in humans (34). However, while rare, non-O157 STEC possessing the stx2e gene, such as O8:H19 (stx2e), has been associated with acute diarrhea (70).

In summary, the current study detected the presence of O157 STEC in raw ground pork, which suggests that O157 STEC in ground pork can occasionally occur. Another hypothesis regarding factors that might contribute to the presence of O157 STEC and non-O157 STEC in ground pork is cross-contamination of pork with meats of other species during processing. Further studies to gather prevalence data at the establishment level would be beneficial. Although the occurrence of non-O157 STEC was found to be higher than O157 STEC in ground pork samples, the virulence gene profiles identified in this study suggest that the non-O157 STEC isolates from ground pork found in this study are not those known to cause severe human illness; however, some may have the potential to cause diarrhea in humans. Finally, education regarding the risk associated with STEC contamination of pork should be considered for the public and those in the food industry to help reduce foodborne illnesses in humans.

We are thankful for Dr. Roger Johnson's input. At the CFIA, we thank all staff involved in the development and implementation of this project. We also thank the staff across the CFIA laboratory network for their technical assistance. This project was supported by the CFIA.

Supplemental material associated with this article can be found online at: https://doi.org/10.4315/JFP-21-147.s1

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This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/)

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