Shiga Toxin Subtypes Associated with Shiga Toxin–Producing Escherichia coli Strains Isolated from Red Deer,Roe Deer,Chamois,and Ibex

Abstract

A total of 52 Shiga toxin–producing Escherichia coli (STEC) strains, isolated from fecal samples of six ibex, 12 chamois, 15 roe deer, and 19 red deer were further characterized by subtyping the stx genes, examining strains for the top nine serogroups and testing for the presence of eae and ehxA. Eleven of the 52 strains belonged to one of the top nine STEC O groups (O26, O45, O91, O103, O111, O113, O121, O145, and O157). Eight STEC strains were of serogroup O145, two strains of serogroup O113, and one strain of serogroup O157. None of the strains harbored stx2a, stx2e, or stx2f. Stx2b (24 strains) and stx1c (21 strains) were the most frequently detected stx subtypes, occurring alone or in combination with another stx subtype. Eight strains harbored stx2g, five strains stx2d, three strains stx1a, two strains stx2c, and one strain stx1d. Stx2g and stx1d were detected in strains not harboring any other stx subtype. The eae and ehxA genes were detected in two and 24 STEC strains, respectively. Considering both, the serogroups and the virulence factors, the majority of the STEC strains isolated from red deer, roe deer, chamois, and ibex do not show the typical patterns of highly pathogenic STEC strains. To assess the potential pathogenicity of STEC for humans, strain isolation and characterization is therefore of central importance.

Introduction

S higa toxin (Stx)–producing Escherichia coli (STEC) is among the most common causes of foodborne diseases (Anonymous, 2011a). This organism is responsible for several human gastrointestinal illnesses, including nonbloody or bloody diarrhea. Especially in children, this disease may be affected by neurologic and renal complications, including hemolytic uremic syndrome (HUS). Most outbreaks and sporadic cases of bloody diarrhea and HUS have been attributed to strains of STEC serotype O157:H7. However, in Europe the role of non-O157 STEC strains (e.g., O26:H11/H–, O91:H21/H, O103:H2, O111:H–, O113:H21, O121:H19, O128:H2/H–, and O145:H28/H–) as a cause of HUS, bloody diarrhea, and other gastrointestinal illnesses is being increasingly recognized (Anonymous, 2011a).

The common feature and main virulence factor of STEC is production of Stx1 and/or Stx2 proteins. Human pathogenic STEC strains often may also harbor other virulence factors such as intimin (eae), a protein essential for the intimate attachment and the formation of attaching and effacing lesions on gastrointestinal epithelial cells, and EHEC-Enterohemolysin (ehxA) (Paton and Paton, 1998).

STEC have been isolated from feces of a variety of healthy domestic and wild animals, but domestic ruminants, especially cattle, are regarded as the principal reservoir of STEC for human infection (Karmali et al., 2010). Nevertheless, wild ruminants may be a potential reservoir and therefore a source for human infections (Asakura et al., 1998; Sanchez et al., 2009; Bardiau et al., 2010; Kistler et al., 2011), and deer meat has been implicated in the transmission of STEC to humans (Keene et al., 1997; Rabatsky-Ehr et al., 2002; Ahn et al., 2009; Rounds et al., 2012). Minimal characterization data are available so far for STEC strains from wild ruminants and also for stx subtypes and virulence factors of non-O157 STEC strains from wild ruminants. Such data, however, are necessary to gain insight into the relationship of these strains and strains isolated from patients. In this study, we characterized strains isolated from red deer, roe deer, chamois, and ibex by (i) subtyping the stx genes, (ii) examining strains for the top nine serogroups (O26, O45, O91, O103, O111, O113, O121, O145, O157), and (iii) testing strains for the presence of eae and ehxA genes.

Methods

Sampling and STEC detection

Hunters collected fecal samples during the hunting season in autumn 2011 in the field, immediately after evisceration. For each sampled animal, sex, age, and location of hunting were recorded. After opening of the large intestine, fecal matter was collected from the colon, placed into sterile tubes, and stored frozen. In total, 239 faecal samples were obtained from three cantons in Switzerland (Graubünden, St. Gallen, Zug). Thereby, 84 samples originated from red deer (Cervus elaphus), 64 from roe deer (Capreolus capreolus), 64 from chamois (Rupicapra rupicapra), and 27 from ibex (Capra ibex), respectively. From each fecal sample about 1 g was enriched in 10 mL modified tryptic soy broth (mTSB, CMO989; Oxoid AG, Pratteln, Switzerland) supplemented with 16 mg/L novobiocin (novobiocin sodium; Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) for 18–24 h at 37°C. From 50 μL of the enrichment broth, a lysate was made (lysis tube; Pall GeneDisc Technologies, Bruz, France), and real-time polymerase chain reaction (PCR) for stx was performed using the commercially available GeneDisc system (Pall GeneDisc Technologies, Bruz, France), following manufacturer's instructions.

Isolation of STEC strains

One loopful from the enrichment broth of each stx-positive sample was plated onto sheep blood agar (Difco^™ Columbia Blood Agar Base EH, Becton Dickinson AG, Allschwil, Switzerland; 5% sheep blood SB055, Oxoid AG) and incubated for 18–24 h at 37°C. Thereafter, five colonies were picked, subcultivated on sheep blood agar for 18–24 h at 37°C, and tested with the LightCycler 2.0 (Roche Diagnostics AG, Rotkreuz, Switzerland) for the presence of stx1 and stx2 group genes by using the primers and probes described by Perelle et al. (2004).

Further characterization of STEC strains

From each fecal sample with stx-positive isolates, one isolate was selected for further strain characterization. The strains were tested for the top nine serogroups (O26, O45, O91, O103, O111, O113, O121, O145, O157) by real-time PCR using GeneDisc system (Pall GeneDisc Technologies). Moreover, STEC strains were examined for the presence of eae and ehxA genes (Schmidt et al., 1995; Møller Nielsen and Thorup Andersen, 2003). Shiga toxin genes were subtyped by PCR according to a standard procedure proposed by the World Health Organization Collaborating Center for Reference and Research on Escherichia and Klebsiella (Anonymous, 2011b). Strains were thereby tested for stx1a, stx1c, and stx1d if stx1-positive and for stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g if stx2-positive in the stx group specific PCR, respectively.

Results

Of the 239 sampled animals, 103 (45%) tested stx positive in the screening PCR. By picking five colonies from each stx-positive sample, 52 STEC strains from 52 different animals were isolated. Six of the strains originated from ibex, 12 from chamois, 15 from roe deer, and 19 from red deer, respectively. The characterization data of the strains for serogroups, stx subtypes, and the presence of eae and ehxA are summarized in Table 1. Eleven of the 52 isolated strains belonged to one of the top nine serogroups. Eight STEC strains were of serogroup O145, two strains of serogroup O113, and one strain of serogroup O157. None of the strains harbored stx2a, stx2e, and stx2f. Stx2b (24 strains) and stx1c (21 strains) were the most frequently detected stx subtypes, occurring alone or in combination with another stx subtype. Eight strains harbored stx2g, five strains stx2d, three strains stx1a, two strains stx2c, and one strain stx1d. Stx2g and stx1d were detected in strains not harboring any other stx subtype. The gene encoding for the outer membrane protein intimin was detected in two STEC strains from red deer. One of these strains was of serogroup O157. Twenty-four STEC strains (46%) tested positive for ehxA. There was no association between stx-subtype, the presence of ehxA, or origin of the strains.

Table 1.

Origin and Characteristics of Shiga Toxin–Producing Escherichia coli (STEC) Strains Isolated from Hunted Wild Ruminants

Number of strains	Origin	Serogroup	stx1-subtype	stx2-subtype	eae	ehxA
3	Chamois	Others^a	stx1c	—	−	+
1	Chamois	Others^a	stx1c	—	−	−
1	Chamois	O145	—	stx2b	−	−
1	Chamois	Others^a	—	stx2b	−	+
4	Chamois	Other^a	stx1c	stx2b	−	+
2	Chamois	O145	stx1c	stx2b	−	−
2	Ibex	Others^a	stx1c	—	−	+
1	Ibex	O113	stx1c	stx2b	−	+
1	Ibex	O145	—	stx2b	−	+
1	Ibex	Others^a	stx1a	stx2b	−	−
1	Ibex	Other^a	stx1c	stx2b	−	+
1	Red deer	Others^a	stx1a	—	+	+
1	Red deer	O113	stx1c	—	−	−
1	Red deer	Others^a	stx1c	—	−	−
3	Red deer	O145	—	stx2b	−	−
2	Red deer	Others^a	—	stx2b	−	+
2	Red deer	Others^a	—	stx2b	−	−
1	Red deer	O157	—	stx2c	+	+
1	Red deer	Others^a	—	stx2c, stx2d	−	+
3	Red deer	Others^a	—	stx2d	−	−
2	Red deer	Others^a	—	stx2g	−	−
2	Red deer	Other^a	stx1c	stx2b	−	+
1	Roe deer	Others^a	stx1c	—	−	+
2	Roe deer	Others^a	stx1c	—	−	−
2	Roe deer	Others^a	stx1d	—	−	−
1	Roe deer	O145	—	stx2b	−	+
2	Roe deer	Others^a	—	stx2b	−	−
5	Roe deer	Others^a	—	stx2g	−	−
1	Roe deer	Others^a	—	stx2g	−	+
1	Roe deer	Others^a	stx1a	stx2d	−	+

Non O26, O45, O91, O103, O111, O113, O121, O145, and O157.

Discussion

STEC strains pathogenic for humans tend to feature Stx2 and other virulence traits such as the adhesion factor intimin (Friedrich et al., 2002; Brooks et al., 2005). In STEC strains characterized in this study, stx2 was the predominant stx gene identified, a result which is in agreement with previous studies on STEC isolates from wild ruminants (Sanchez et al., 2009; Bardiau et al., 2010; Kistler et al., 2011) or isolates from game meat (Miko et al., 2009). With respect to the stx subtypes (according to the established new Stx nomenclature) (Persson et al., 2007), stx2b was the predominant variant among the STEC isolates in the present study. Subtype stx2b has also frequently been reported in STEC strains from deer meat (Miko et al., 2009), and this variant most likely does not cause severe human diseases, since it is mainly found in strains isolated from healthy human carriers (Stephan and Hoelzle, 2000). Eight strains harbored stx2g, a stx variant which was originally described in a bovine fecal sample and which seems to have a minor role in human infections (Prager et al., 2011). Moreover, stx1c-harboring strains, which were frequently found in the STEC from wild game, are associated with asymptomatic human carriage or mild disease (Friedrich et al., 2003). Nevertheless, two of the isolates (one strain belonging to the O157 serogroup and one strain not belonging to the top nine serogroups) in the present study carried subtype stx2c, which has been associated with high virulence, and strains producing this stx subtype have been isolated from patients with hemolytic colitis and HUS (Friedrich et al., 2002; Persson et al., 2007; Käppeli et al., 2011). Moreover, four other strains, isolated from three red deer and one roe deer, harbored stx2d. The Stx2d type also belongs to the group of Shiga toxin genes in the Stx2acd group (stx2a, stx2c, stx2d), which are genetically closely related and are reported to be associated with HUS in patients (Friedrich et al., 2002; Persson et al., 2007). The gene encoding for outer membrane protein intimin (eae) was detected only in two (one strain belonging to the O157 serogroup and one strain not belonging to the top nine serogroups) of the 52 STEC strains. In contrast the ehxA gene was found in high prevalence (46%). These results are in agreement with previous studies on STEC isolates from wild ruminants (Gilbreath et al., 2009; Sánchez et al., 2009; Bardiau et al., 2010).

In summary, STEC can be detected in high frequency (45%) in fecal samples of wild ruminants. Considering both, the serogroups and the virulence factors, the majority of the STEC strains isolated from red deer, roe deer, chamois, and ibex do not show the typical patterns of highly pathogenic STEC strains. Nevertheless, highly pathogenic strains can be found. Therefore, to assess the potential pathogenicity of STEC strains from wild ruminants for humans, strain isolation and characterization are therefore of central importance.

Footnotes

Disclosure Statement

No competing financial interests exist.

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