Characterization of Shiga Toxin Subtypes and Virulence Genes in Porcine Shiga Toxin-Producing Escherichia coli

Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.


INTRODUCTION
Shiga Toxin-producing Escherichia coli (STEC) are food-borne pathogens responsible for outbreaks and serious illness including hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). STEC O157:H7 is the serotype that has most often been associated with outbreaks and severe forms of diarrhea; however, recently a number of non-O157 STEC serogroups that cause similar illnesses have emerged (Gould et al., 2013). Cattle and other ruminants are important reservoirs of STEC; infection is asymptomatic, and they can carry the pathogens for long periods of time. Similarly, healthy swine may shed STEC, as demonstrated by several studies in which STEC were detected and isolated from swine fecal samples (Tseng et al., 2014b). Many of the investigations focused on serotype O157:H7; however, some studies also tested for non-O157 STEC serogroups and identified serogroups previously associated with human cases of illness (Fratamico et al., 2004;Kaufmann et al., 2006;Tseng et al., 2014b). The possibility that swine can transmit pathogenic STEC to humans is supported by a few outbreaks linked to the consumption of pork products contaminated with STEC O157:H7, O157:NM, and O111:H- (Tseng et al., 2014b).
Shiga toxins (Stx) are divided in two major antigenic forms: Stx1 and Stx2. Variants for Stx1 and Stx2 are grouped in three (Stx1a, Stx1c, Stx1d) and seven (Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g) subtypes, respectively (Scheutz et al., 2012). Although Stx1a has been linked to human illness, STEC that produce subtypes Stx2a, Stx2c, and Stx2d are more often associated with the development of HC and HUS (Friedrich et al., 2002;Melton-Celsa, 2014). In vitro studies in two different cell lines showed that Stx2a and Stx2d were more potent than Stx2b and Stx2c. These results were also confirmed by experimentation in mice showing a significantly higher potency of Stx2a and Stx2d than Stx1, Stx2b, and Stx2c (Fuller et al., 2011). Stx variants are not homogeneously distributed among the STEC population and certain variants are frequently detected in association with different animals Hofer et al., 2012;Fuente et al., 2015). Swine STEC strains commonly produce Stx2e (Fratamico et al., 2004;Meng et al., 2014;Tseng et al., 2015), which may cause edema disease in weaned pigs, often leading to ataxia and death. Stx2e-producing Escherichia coli, do not represent a particular threat for humans (Friedrich et al., 2002;Tseng et al., 2014b). Nevertheless, STEC carrying the stx 2e gene have been isolated from human cases with mild diarrhea (Muniesa et al., 2000;Friedrich et al., 2002;Beutin et al., 2004;Sonntag et al., 2005) and from two patients with HUS (Thomas et al., 1994;Fasel et al., 2014). The severe outcome of the first HUS case was probably due to a co-infection with another STEC strain (Thomas et al., 1994), while the second patient with HUS was described as having a very weak immune system (Fasel et al., 2014). Besides Stx2e, there is a lack of information on the presence of other Stx subtypes in STEC strains isolated from swine.
The production of Stx is necessary to provoke HUS; however, other virulence factors are also important in causing illness. These include genes involved in cell adhesion, proteases, and toxins, as well as other putative virulence factors. The presence of specific combinations of virulence factors may determine the risk of developing severe symptoms. The eae gene, found on the locus of enterocyte effacement (LEE), encodes intimin, which is an adhesin involved in gut colonization. LEE-positive STEC are expected to provoke HUS or HC more frequently than LEE-negative STEC (Ethelberg et al., 2004;Toma et al., 2004;Luna-Gierke et al., 2014). Nevertheless, cases of HUS provoked by LEE-negative STEC have been reported (Karmali et al., 1985;Paton et al., 1999;Bielaszewska et al., 2009), including a large outbreak in 2011 in Europe caused by an enteroaggregative E. coli that acquired the stx 2a gene, and it possessed a combination of virulence genes increasing its virulence (Boisen et al., 2015). This suggests that LEE is not essential in the development of severe symptoms, and other genes involved in adherence may also be important. Many adherence gene candidates, including eibG, lpfA, saa, and sab have been identified in STEC (Croxen et al., 2013). Nevertheless, mechanisms for attachment of LEE-negative STEC to the intestinal epithelium have not been studied as extensively as attachment of LEE-positive STEC.
In 2000, one objective of the U.S. Department of Agriculture's Animal and Plant Health Inspection Service National Animal Health Monitoring System (NAHMS) Swine 2000 study was to determine the prevalence of STEC in swine. Fecal samples were from states with the highest production of swine in the U.S. (U.S. Department of Agriculture, 2001). As a result of this work, 219 STEC isolates were recovered and characterized (Fratamico et al., 2004(Fratamico et al., , 2008. Since this work was conducted, the knowledge of the importance of non-O157 STEC in human illness has increased, and there is a need to develop a model for molecular risk assessment associated with STEC. Knowledge of the virulence gene combinations that distinguish highly pathogenic E. coli from less virulent strains remains unclear, particularly for LEE-negative STEC (Beutin and Fach, 2014). Additionally, new virulence-associated and putative virulenceassociated factors are being identified (Coombes et al., 2008;Brandt et al., 2011;Bugarel et al., 2011). The aim of the present study was to characterize STEC recovered from swine, belonging to a variety of serotypes to determine their Stx subtype and virulence gene profiles to understand their virulence potential.

Bacterial Strains
Swine STEC strains were isolated and serotyped during the NAHMS swine 2000 study (NAHMS 2000) as described by Fratamico et al. (2004). Briefly, fresh swine feces were recovered from the pen floor of swine operations from the main porkproducing states in U.S. A total of 687 swine fecal samples were enriched using tryptic soy broth (TSB) and screened for the presence of stx 1 and stx 2 by polymerase chain reaction (PCR). Positive samples were plated onto Luria-Bertani agar, and stx 1 -and stx 2 -positive colonies were detected following DNA hybridization and confirmed by PCR. Two hundred and nineteen STEC strains were serotyped and frozen in TSB with 20% of glycerol. From this collection, 181 STEC strains were used in this study and maintained on tryptic soy agar plates or TSB as working stock cultures.
Besides the NAHMS swine isolates, three STEC O91 strains from our collection were also used for comparison. STEC O91:H14 (strains 2.4111 and 2.4114) were isolated from ground beef while STEC O91:H21 (strain B2F1) was isolated from a case of HUS (Ito et al., 1990).

Identification of Shiga-toxin Subtypes
DNA extraction and PCR assays to identify stx subtypes and stx partial sequences were performed according to Scheutz et al. (2012) using a ProFlex PCR system (Thermo Fisher, Waltham, MA, USA) with slight modifications. TaqMan Environmental Master Mix 2.0 (Thermo Fisher) was used, and the annealing temperature was raised to 65 • C when cross-reaction was observed, as suggested by the authors (Scheutz et al., 2012). Gel electrophoresis was performed using 1.5% UltraPure Agarose (Invitrogen, Carlsbad, CA, USA) gel with 0.5X GelRed (Phenix Research Products, Candler, NC, USA) in 1X Tris-acetate-EDTA buffer at 100 V for 1 h. One microliter of amplified DNA was analyzed by agarose gel electrophoresis and visualized using an AlphaImager gel documentation system (Alpha Innotech, San Leandro, CA, USA).

FDA Minimal Signature E. coli Array
Swine Stx2d-producing E. coli and non-Stx2e STEC belonging to a serotype associated with human disease were further analyzed using the Minimal Signature E. coli Array Strip (FDA-ECID; Affymetrix, Santa Clara, CA, USA). Genomic DNA was isolated and concentrated using the DNeasy Tissue Kit (QIAgen Inc., Valencia, CA, USA) and SC100 Speedvac Concentrator (Savant Instruments, Inc. Holbrook, NY, USA), respectively. Two micrograms of DNA were tested using the FDA-ECID array as described in detail by Lacher et al. (2014). Robust multiarray average summarized probe intensity data were analyzed using R-Bioconductor software v3.1.2 and affy package with parameters defined by Lacher et al. (2014). The Hierarchical clustering was done using overview function in MADE4 package that uses average linkage cluster analysis with a correlation metric distance (Culhane et al., 2005;Culhane and Thioulouse, 2006).

Swine STEC Serotypes
All of the strains had been previously serotyped at the E. coli Reference Center at the Pennsylvania State University (University Park, PA, USA). In addition, many O-group-and H-groupspecific targets were included in the hrPCR assay. Several discrepancies were found and serotypes that did not match with the traditional serotyping are indicated in bold in Figure 1. Selected swine STEC strains were also analyzed using the FDA-ECID microarray, and the resulting serotypes were in agreement with the hrPCR. Moreover, the grouping within the phylogenetic tree was consistent with the serotypes proposed by the FDA-ECID microarray ( Table 1).

DISCUSSION
It is well-known that swine shed a variety of STEC serogroups, which may be carried along the food production chain. Most of the STEC isolated from these animals have adapted to the swine host and seem to have low potential to infect humans. Nevertheless, outbreaks associated with pork products have occurred (Meng et al., 2014;Tseng et al., 2014b). The sampling area covered by the NAHMS swine 2000 study was large, covering all the main pork-producing States (Fratamico et al., 2004). A subset of 181 STEC strains were analyzed and their pathogenic potential was assessed by detection of virulence and putative virulence factors.
The stx subtypes carried by the swine STEC were identified, and the majority of the isolates carried stx 2e (81%), which was consistent with the data reported by Fratamico et al. (2004). The second most prevalent subtype was stx 1a (14%), followed by stx 2d (3%), and stx 1c (1%). Stx2d is a potent toxin, and infection with strains carrying this subtype can lead to severe symptoms such as HC and HUS in humans (Melton-Celsa, 2014). Besides the Stx genes, the thermostable enterotoxin genes, astA and/or estIa, genes were found in ∼71% of the isolates. Thermostable enterotoxins are usually carried by enterotoxigenic E. coli, which are the major pathogens responsible for traveler's diarrhea. Twenty-two percent of the swine STEC strains were positive for both genes. The exotoxins HlyA (α-hemolysin) and EhxA (enterohemolysin) produce pores in the cytoplasmic membranes of eukaryotic cells causing their death. Their role in STEC pathogenesis is still not clear; HlyA may increase the virulence of extraintestinal pathogenic E. coli and, in the case of EhxA, a correlation between ehxA-positive STEC and development of severe symptoms in humans has been observed (Karch and Bielaszewska, 2001;Mainil, 2013). Thirteen isolates carried the hlyA gene. Nine of them belonged to serotypes O121:H-or O121:H10, presenting a virulence gene profile typical of strains associated with edema disease in swine due to the presence of stx 2e , hlyA and fedA (Tseng et al., 2014b). The ehxA gene is commonly found in STEC.
From 40 to 77% of strains collected from patients, food, and cattle carry this gene (Karch and Bielaszewska, 2001;Slanec et al., 2009;Bosilevac and Koohmaraie, 2011;Feng, 2014). Swine isolates appear to carry ehxA less frequently (Meng et al., 2014;Tseng et al., 2014a), and this observation is in agreement with our study where only 7% of the isolates was ehxA positive.
All of the swine STEC strains were LEE-negative. Although the adhesion mechanisms of LEE-negative STEC are not well characterized, several factors have been described to play an important role in adhesion to the intestinal epithelium. The long polar fimbriae gene lpfA O113 was identified in STEC O113:H21 (Doughty et al., 2002). These investigators demonstrated that the removal of lpfA O113 reduces the bacterial capacity to adhere to epithelial cells. Similar lpfA genes were found in E. coli O157 and O26 (Hayashi et al., 2001;Toma et al., 2004). Another bacterial adherence-conferring gene is the ironregulated gene A homolog adhesin iha. Similarly to lpfA O113 , the iha gene is commonly found in STEC strains associated with human cases of HUS (Newton et al., 2009;Galli et al., 2010). Nevertheless, non-pathogenic E. coli can also carry lpfA O113 and iha, suggesting that the presence of these genes is insufficient to establish an infection (Toma et al., 2004). Over 80% of the strains analyzed in this study carried lpfA O26 , lpfA O113 , or lpfA O157 ; while iha was found in almost one quarter of swine isolates. iha-positive STEC were also described in a longitudinal study of two Midwestern U.S. pork production sites (Tseng et al., 2014a(Tseng et al., , 2015. On the contrary, none of the swine STEC strains collected in another interesting study in China carried iha (Meng et al., 2014). The second most prevalent adhesion factor found in this dataset was the porcine attaching and effacing-associated adhesin, paa, which is associated with neonatal post-weaning diarrhea in pigs . In addition, a few strains carried orfA and orfB, which encode for adhesins involved in diffuse adherence (Charbonneau et al., 2006).
Autotransporter proteins have a peculiar structure that allows them to move through the membrane system and execute their function outside the bacterial cell. The genes ehaA and sab were discovered in O157:H7 strain EDL933 and LEEnegative O113:H21, respectively. They encode for two different autotransporter proteins that contribute to adhesion and biofilm formation (Wells et al., 2008;Herold et al., 2009). Together with LEE genes, iha and ehaA are highly expressed in the intestines of pigs presenting attaching and effacing lesions (Liu et al., 2015). While the ehaA gene was present in over 30% of the swine isolates, sab was carried by 13 STEC strains only belonging to O-group O91.
As stated above, 12 to 18% of the isolates were positive for katP, ureD, and terE. The genes katP and ureD encode for a catalase/peroxidase and urease transporter, respectively. Their role in E. coli pathogenesis is unclear; however, they appear to be prevalent in diarrheagenic E. coli (Dorothea et al., 2006;Delannoy et al., 2013). The gene terE is a component of the ter cluster, which confers tellurite resistance (Orth et al., 2007). The ecs1763 and ecs1822 genes have been proposed to be novel markers for enterohemorrhagic E. coli. Their function is unknown, and they were shown to be shared by a clonal group of enterohemorrhagic E. coli that includes O26, O111, and O118 (Abu-Ali et al., 2009). Tseng et al. (2014a) observed that ecs1763 is frequently found in swine STEC, which was confirmed by the present study where 31% of the isolates carried ecs1763. ecs1822 was absent in all the tested strains.
Shiga toxin-producing Escherichia coli strain 308 was re-typed as O15:H27 using the FDA-ECID array and was found to have the same stx 2d sequence as E. coli O15:H27 (strain 88-1509) in the STEC isolate database at Michigan State University 2 . E. coli strain 88-1509 was collected in 1988 from a human case of HC and HUS in Canada. Other strains belonging to serotype O15:H27 have been isolated from human and cattle feces, and from meat sources (Piérard et al., 1997;Woodward et al., 2002;Bosilevac et al., 2007;Galli et al., 2010). The LEE-negative swine STEC O15:H27 has a virulence gene profile consisting of stx 1c , stx 2d , ehaA, espP, fyuA, ihA, irp2, lpfA O113 , and Z2099. The relevance of some of these genes was mentioned above. E. coli secreted protein P (EspP) is an autotransporter protein with serine protease activity, and is used by the bacteria to impair the complement response of the host (Orth et al., 2010). Recently, In et al. (2013) reported that EspP boosts macropinocytosis in the intestinal epithelium increasing Stx uptake. The open reading frame Z2099 is highly prevalent in typical and emerging enterohemorrhagic E. coli, while it is significantly less prevalent in non-pathogenic E. coli .
Six of the swine STEC strains carried stx 2d according to PCR and VirulenceFinder results, and they belonged to serotypes O159:H-, O159:H4, and OX10:H-. DebRoy et al. (2016) reported that serological cross-reactions between the O159 and OX10 O-groups often occur and that the nucleotide sequences of O159 and OX10 O-antigen gene clusters are almost identical. Based on the FDA-ECID analysis, the strains 306, 360, 341, and 500 were re-typed as O159:H16; while the strain 326 was re-typed as O159:H-( Table 1). STEC belonging to O-group O159 rarely infect humans (Brooks et al., 2005;Gould et al., 2013). STEC O159:H16 and O159:H-have been isolated only from swine samples, such as feces and carcasses (DesRosiers et al., 2001;Kaufmann et al., 2006;Meng et al., 2014). Stx subtype analysis of these strains often gives ambiguous results (Kaufmann et al., 2006;Meng et al., 2014). In this work, STEC O159:H16 and O159:H-were positive for stx 2d when tested by PCR; however, they were positive for stx 2e or stx 2i using the FDA-ECID array. Note that probes of the FDA-ECID array corresponding to stx 2i were designed using the Stx sequences AM904726 and FN252457 (Patel et al., 2016) that belong to the stx 2e subtype according to Scheutz et al. (2012). The product obtained from partial sequencing of stx 2 was 99% identical to the sequence KC339670 when blasted against the NCBI database. KC339670 is a complete stx 2 sequence belonging to a STEC O159:H16 strain isolated from swine in China. After a neighborjoining cluster analysis of the sequence, Meng et al. (2014) concluded that KC339670 represented a new variant of stx 2e . Further investigations using cell lines and animal models are needed to understand the virulence potential of this Stx2 variant. Another STEC O159 was detected in this collection. It belonged to the H21 H-group and was positioned distantly from the clade of O159:H16 and O159:H-( Table 1). This strain was positive for stx 2e only by PCR. E. coli belonging to serotype O159:H21 was isolated in 1983 during a small outbreak of diarrhea involving newborn children in Spain (Blanco et al., 1992), and no other infections associated with serotype O159:H21 have been reported.
Locus of enterocyte effacement-negative STEC belonging to O-group O91 are frequently associated with adult human infections with symptoms ranging from mild diarrhea to HC and HUS. The main serotypes are O91:H14 and O91:H21, and the latter is usually linked with development of severe symptoms (Bielaszewska et al., 2009). Human STEC O91:H14 and O91:H21 isolates carried mainly stx 1 and stx 2d , respectively (Prager et al., 2005;Bielaszewska et al., 2009;Galli et al., 2010). These STEC have been isolated from food samples derived from bovine, swine, and ovine origin, and from both domestic and wild animals Ju et al., 2012). From the NAHMS swine 2000 study, 15 strains belonging to serotypes O91:H12, O91:H14, O91:H44, and O91:H-were isolated from fresh fecal samples collected from four different states (Fratamico et al., 2004). Eight of these strains were re-typed as O91:H14, while STEC O91:H44 strains 448 and 477 did not belong to the O91 O-group based on FDA-ECID and hrPCR results (Figure 1; Table 1). STEC strain 319 that carried an identical virulence gene profile to other O91:H14 strains was also re-typed as O91:H14 by FDA-ECID array ( Table 1). According to the phylogenetic tree in Table 1, the clade of STEC O91:H14 strains is well separated from the STEC O91:H21 strain B2F1 isolated from a human case of HUS. Interestingly, two O91:H14 strains were more closely related to two STEC O91:H14 strains isolated from ground beef samples than the other swine STEC O91:H14 strains. Despite the fact that one strain was katP-negative, all 13 STEC O91:H14 strains presented a conserved virulence gene profile (ehaA, ehxA, eibG, espP, ihaA, katP, lpfA O26 , lpfA O113 , pagC, sab, and stx 1a ), which is very similar to profiles of strains from human clinical samples (Prager et al., 2005;Bielaszewska et al., 2009). Similar to ihaA, lpfA O26 , and lpfA O113 , the proteins encoded by the genes eibG and sab are involved in host gut colonization. The E. coli immunoglobulin-binding protein encoded by eibG binds human immunoglobulin G and immunoglobulin A, and contributes to epithelial host cell adhesion (Lu et al., 2006), and sab is a gene encoding for an autotransporter protein involved in biofilm formation and found in a pathogenic LEE-negative STEC (Herold et al., 2009). Lastly, the pagC gene encodes for an outer membrane protein present in different Enterobacteriaceae that contributes to serum resistance (Nishio et al., 2005).
STEC O20:H19 is associated with human cases of HUS (Galli et al., 2010), and one strain belonging to this serotype was isolated in the NAHMS study (Fratamico et al., 2004). However, this same strain was re-analyzed using the FDA-ECID array, and it was retyped as O152:H19, which is not known to be a human pathogen.

CONCLUSION
Using state-of-the-art DNA-based techniques, this study provides new insights on the distribution of virulence factors in a heterogeneous collection of STEC isolated from the major porkproducing states of the U.S. Stx2e-producing E. coli known to provoke mild diarrhea in humans carried different virulence factors than Stx2e-producing E. coli associated with edema disease in pigs; this finding suggests that Stx2e-producing E. coli that cause human illnesses may not have a swine origin (Sonntag et al., 2005). In our work, STEC strains carrying stx 2e belonging to the same serotype and having similar virulence gene profiles as Stx2e-producing E. coli isolated from humans were identified. Additionally, the majority of Stx2e-producing E. coli carried thermostable enterotoxin genes usually found in enterotoxigenic E. coli.
This work suggests that STEC, including serotypes O15:H27 and O91:H14 that have been associated with human illness and are found in multiple hosts or environments, could also be carried by swine. Interestingly, a strain of O15:H27 found to carry stx 2d and other virulence genes may have the potential to produce severe symptoms in humans. Moreover, STEC O91:H14 strains presented a virulence gene profile very similar to profiles found in human isolates.

AUTHOR CONTRIBUTIONS
GMB and PF. designed research; GMB, LKB, SD, PF, FB, AA, and TP performed research; GMB, JG, and IP analyzed data; GMB and PF wrote the paper.

ACKNOWLEDGMENTS
This research was supported in part by an appointment to the Agricultural Research services (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the USDA. ORISE is managed by ORAU under DOE contract number DE-AC05-06OR23100. The hrPCR development was partially financed by the French joint ministerial program of R&D against CBRNE risks. All opinions expressed in this manuscript are the authors' and do not necessarily reflect the policies and views of USDA, ARS, DOE, or ORAU/ORISE.