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Journal of Clinical Microbiology, May 2001, p. 1932-1937, Vol. 39, No. 5
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.5.1932-1937.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

The Common Ovine Shiga Toxin 2-Containing Escherichia coli Serotypes and Human Isolates of the Same Serotypes Possess a Stx2d Toxin Type

Vidiya Ramachandran,^1,2 Michael A. Hornitzky,¹ Karl A. Bettelheim,³ Mark J. Walker,² and Steven P. Djordjevic^1,*

Elizabeth Macarthur Agricultural Institute, Camden, New South Wales 2570,¹ Department of Biological Sciences, University of Wollongong, New South Wales 2522,² and Microbiological Diagnostic Unit, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3052,³ Australia

Received 21 September 2000/Returned for modification 9 January 2001/Accepted 8 March 2001

	ABSTRACT

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Shiga toxin 2 (Stx2) has been reported as the main Shiga toxin associated with human disease. In addition, the Stx2 toxin type can have a profound impact on the degree of tissue damage in animal models. We have characterized the stx₂ subtype of 168 Shiga toxin-producing Escherichia coli (STEC) isolates of which 146 were derived from ovine sources (principally feces and meat) and 22 were isolated from humans. The ovine STEC isolates were of serotypes that have been shown to occur commonly in the gastrointestinal tract of healthy sheep. The major stx₂ subtype in the ovine isolates was shown to be stx_2d-Ount (119 of 146 [81.5%]) and was predominantly associated with serotypes O75:H/H8/H40, O91:H, O123:H, O128:H2, and OR:H2. However, 17 of 18 (94.4%) ovine isolates of serotype O5:H possessed a stx_2d-O111/OX3a subtype. Furthermore, STEC isolates of serotypes commonly found in sheep and recovered from both clinical and nonclinical human infections also contained a stx_2d (stx_{2d-Ount/O111/OX3a}) subtype. These studies suggest that a specific stx₂ subtype(s) associates with serotype and may have important epidemiological implications for tracing sources of E. coli during outbreaks of STEC-associated diseases in humans.

	INTRODUCTION

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Multiple virulence factors contribute to the pathogenicity of Shiga toxin-producing Escherichia coli (STEC). Although Shiga toxins are the primary virulence factor, the ability to produce intimin (encoded by eaeA) and the possession of a plasmid encoding enterohemolysin (ehxA) are also important (2, 5, 11, 14). Shiga toxins comprise two immunologically non-cross-reactive groups designated Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Stx1 is virtually identical to the Shiga toxin of Shigella dysenteriae (21). Stx2 is considered to be the most important virulence factor associated with human disease (5, 22). In addition, Stx2 is about 400-fold more toxic to mice than Stx1 and has also been shown to induce fetoplacental resorption, intrauterine hematoma, fibrin deposition, and neutrophil infiltration when injected intravenously into mice on day 5 of pregnancy (32, 34). Unlike for stx₁, considerable sequence variation among stx₂ genes has been reported (12, 26, 30, 33). More importantly, differences in the degree of pathogenicity of STEC serotypes have been associated with variations in the stx₂ subtype (13, 16, 17).

At least 10 stx₂ gene variants have been described (10, 12, 19, 24, 25, 26, 29, 30, 33). The most prevalent Stx2 variants are stx_2c, stx_2d, and stx_2e (26, 30, 33). stx_c was isolated from E. coli O157:H strain E32511 and is closely related to stx₂ and stx_2vha (30). The stx_2d cluster as defined by Pierard et al. (26) comprises stx_2d-O111 (24), stx_2d-OX3a (25), and stx_2d-Ount variants, and these subtypes were identified in non-O157 STEC strains isolated from humans and meat (26, 27). However, stx_2d-positive STEC strains are not observed in the most virulent serogroups for humans, including O157, O26, O103, O111, and O145 and have been reported to be less frequently associated with diarrhea and hemolytic uremic syndrome (HUS) (26). stx_2e is predominantly associated with edema disease in swine (33) and is rarely recovered from humans.

The importance of characterizing Stx2 types has been recently highlighted by the observation that mouse or human colonic mucin (18) can activate some Stx2 toxins. The Vero cell cytotoxicity of intestinal mucus-treated Stx2vha/b was reported to increase 35- to 350-fold compared to non-mucin-treated Stx2vha/b. Mucin activation provides an explanation for the observation that STEC strains expressing Stx2vh are highly virulent (50% lethal dose of <10 CFU) when fed to streptomycin-treated CD-1 mice compared to STEC strains expressing Stx2c (50% lethal dose of 10¹⁰ CFU) (16, 17).

Recent studies of sheep in eastern Australia have demonstrated that the predominant STEC serotypes containing accessory virulence factors (enterohemolysin and/or intimin) are O5:H, O75:H8, O91:H, O123:H, and O128:H2 (6a), and several of these serotypes have been occasionally isolated from clinically affected patients. More than 60 different serotypes of STEC have been isolated from humans with clinical infections (1). Many STEC isolates of ovine origin contain stx₂ and express toxin (6a). However, only a few reports have examined stx₂ subtypes among STEC isolates recovered from ruminant sources, particularly sheep. The aims of this study were (i) to determine the stx₂ subtype(s) of STEC isolates derived from ovine sources and (ii) to determine the stx₂ subtypes among human STEC isolates that possess a serotype commonly associated with sheep, with the purpose of determining if sheep represent a source of STEC for human infections.

	MATERIALS AND METHODS

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STEC isolates. One hundred sixty-eight STEC isolates were used in this study (Table 1). The Elizabeth Macarthur Agricultural Institute (New South Wales, Australia) provided 124 isolates, which were isolated using methods described by Djordjevic et al. (6a). Of these, 121 were isolated from healthy sheep and 3 were isolated from diagnostic submissions in which STEC was not necessarily implicated as the cause of the disease. Thirty-four isolates were obtained from the Victorian Infectious Diseases Laboratory (Melbourne, Australia). These consisted of 11 isolates of human origin, 9 isolates from lamb meat, 2 isolates from sheep feces, 1 isolate from a meat sausage, and 10 isolates from lamb carcasses. Andre Burnens from the National Reference Laboratory for Foodborne Diseases (Bern, Switzerland) provided 10 human isolates from patients with diarrhea or HUS (6, 7). The Swiss isolates possessed serotypes not commonly found in STEC isolates recovered from ovine sources and were included in this study for comparative purposes only. The Swiss isolates were serotyped by Kim Ziebel and Roger Johnson from the Guelph Laboratory, Health Canada, Guelph, Ontario, Canada.

View this table: [in this window] [in a new window]   TABLE 1.   Virulence factor profiles and stx2 subtypes of ovine and human STEC isolates

Multiplex PCR analysis of STEC isolates. All isolates were prepared and subjected to multiplex PCR for the detection of STEC virulence factors stx₁, stx₂, ehxA, and eaeA as described by Paton and Paton (23), with the following modification. For DNA preparation, Instagene matrix (Bio-Rad, Richmond, Calif.) was used as described by Fagan et al. (8). Amplified DNA fragments were resolved by gel electrophoresis (28) using 2% (wt/vol) agarose. Gels were stained with ethidium bromide, visualized with UV illumination, and imaged using a GelDoc 1000 image analysis station (Bio-Rad).

stx₂ subtyping. Ovine and human STEC isolates (Table 1) containing stx₂ were subjected to stx₂ subtyping as described by Pierard et al. (26) and Bastian et al. (3). The primer sequences used are listed in Table 2. For this report stx_2d (stx_2d-Ount, stx_2d-O111, and stx_2d-OX3a) is defined as a nucleotide sequence variant of stx₂ as described by Peirard et al. (26) and does not refer to the mucin-inducible Stx2d toxin subtype (encoded by stx_2vha and stx_2vhb) as defined by Melton-Celsa et al. (17).

View this table: [in this window] [in a new window]   TABLE 2.   Primers used to amplify stx2

stx₂ amplified with VT2-e and VT2-f primers (Table 2) was subjected to restriction endonuclease digestion with HaeIII and PvuII as described by Pierard et al. (26). The PCR product obtained with the LinF and LinR primers (Table 2) was digested with HincII and AccI as described by Bastian et al. (3). PCR products (10 µl) were incubated with 5 U of appropriate enzyme in the buffer provided by the manufacturer. Restriction fragments were separated by agarose gel electrophoresis. stx₂ subtypes were identified based on their restriction profiles (Table 3).

View this table: [in this window] [in a new window]   TABLE 3.   Sizes of restriction fragments used for restriction fragment length polymorphism analysis of stx2

stx₂ DNA sequence analysis. An O91:H isolate (isolate 122-A1) was chosen as a source of stx₂ for sequencing studies for the following reasons. Firstly, O91:H is the most common ovine STEC serotype recovered from Australian sheep. Secondly, the stx_2d restriction fragment length polymorphism profile indicated that it possessed a stx_2d-Ount subtype, which is the most common stx₂ subtype observed among STEC isolates from the feces of Australian sheep. The A and B subunits of stx₂ from isolate 122-A1 were amplified using oligonucleotide primers Stx2F and Stx2R (Table 2). PCRs were carried out in a 50-µl total volume containing 5 µl of nucleic acid (extracted with Instagene matrix) from the isolate, 10 mM Tris-HCl (pH 8.3), 10 mM KCl, 2 mM MgCl₂, 10 pmol of each primer, 200 µM (each) deoxynucleotide triphosphates, and 1 U of Taq DNA polymerase. After an initial denaturing step of 5 min at 95°C, the samples were subjected to 35 cycles of denaturation (95°C, 30 s), annealing (60°C, 45 s), and extension (72°C, 90 s), followed by a single final extension step of 5 min at 72°C. PCR products were analyzed by agarose gel electrophoresis and purified using a QIAquick DNA purification kit (Qiagen, Hilden, Germany). Primers used for sequencing are listed in Table 2. DNA sequence reactions were performed using the Big Dye terminator cycle sequencing ready reaction DNA sequencing kit and electrophoresed on an ABI prism 377 DNA sequencer (Perkin-Elmer, Santa Clara, Calif.). Compilation and analysis of DNA sequence data were performed using Auto Assembler software (Perkin-Elmer). Nucleotide and amino acid homology analysis was performed using the Blast program located on the Australian National Genomic Information Service website (http://www.angis.org.au).

Nucleotide sequence accession number. The sequence of stx₂ from the ovine O91:H isolate (122-A1) has been submitted to the GenBank database under the accession no. AF298816.

	RESULTS

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Detection of STEC virulence factors using multiplex PCR. All 146 ovine STEC isolates contained stx₂. Of these, 143 (97.9%) contained stx₁ and stx₂, 139 (95.2%) contained stx₁, stx₂, and ehxA, and 3 (2%) contained all four virulence factors. All 22 human STEC isolates contained stx₂. Eleven (50%) of these contained stx₁ and stx₂, three (13.6%) contained stx₂, ehxA, and eaeA, and none contained all four virulence factors. The virulence factor profiles for all isolates are presented in Table 1.

Subtyping of stx₂. The most common stx₂ subtype observed among STEC isolates from sheep was stx_2d-Ount (Fig. 1; Table 3). Specifically, 55 of 58 (94.8%) O91:H, 16 of 16 (100%) O75:H8, 24 of 25 (96%) O123:H, 12 of 12 (100%) O128:H2, and 4 of 4 (100%) OR:H2 STEC isolates from sheep contained stx_2d-Ount. Seventeen of 18 (94.4%) O5:H, 3 of 58 (5.1%) O91:H, and 1 of 25 (4%) O123:H STEC isolates from sheep were found to contain either stx_2d-O111 or stx_2d-OX3a. These latter two stx₂ variants were not differentiated due to their high nucleotide sequence homology (99%). Of the 10 human isolates with serotypes commonly isolated from sheep, 9 (90%) also contained stx_2d-Ount. The human O5:H isolate contained stx_2d-O111 and/or stx_2d-OX3a. The four ovine O157:H/H21 isolates possessed stx_2vha. Other human isolates with serotypes not commonly found in sheep contained either stx₂ or stx_2vhb variants (Fig. 2; Table 3). One strain of serotype O91:H21 from a human source contained stx₂ in combination with stx_2vhb. The stx₂ variant in the human strain with serotype O15:H was untypable.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   HaeIII (A) and PvuII (B) digests of PCR products obtained with VT2-e and VT2-f primers. Lanes: M, 100-bp Plus marker; 1, O91:H (ovine); 2, O123:H (ovine); 3, O128:H2 (ovine); 4, O75:H8 (ovine); 5, O5:H (ovine); 6, O91:H (human); 7, O123:H (human); 8, O128:H2 (human); 9, O5:H (human); 10, OX3:H8 (human); 11, O91:H10 (human); 12, O91:H21 (human); 13, O121:H19 (human); 14, O145:H (human); 15, O8:H14 (human).

View larger version (29K): [in this window] [in a new window]   FIG. 2.   HincII (A) and AccI (B) digests of PCR products obtained with LinF and LinR primers. Lanes: M, 100-bp Plus marker; uc, undigested PCR product; 1, O121:H19 (human); 2, O15:H (human); 3, O145:H (human); 4, O7:H (human); 5, O145:H (human); 6, O26:H (human); 7, O121:H19 (human); 8, O26:H (human); 9, O8:H14 (human); 10, O26:H11 (negative control: human isolate with stx1 only); 11, O111:H8 (positive stx2 control; human isolate with both stx1 and stx2); 12, O91:H (ovine); 13, O5:H (ovine).

stx₂ sequence analysis. DNA sequence analysis of stx₂ from the O91:H (122-A1) isolate showed 99% homology with stx_2d-Ount (GenBank accession no. AF043627). The stx₂ DNA sequence was also highly homologous (97%) to stx_2d-OX3a (accession no. X65949) and stx_2d-O111 (accession no. L11078). These stx₂ variants are grouped together as stx_2d as described by Pierard et al. (26).

	DISCUSSION

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Although STEC may contain at least four well-characterized virulence factors (Stx1, Stx2, intimin, and enterohemolysin), Stx2 is considered the most important factor affecting human health (5, 22, 34). In this study the stx₂ subtypes of 146 STEC isolates from sheep and 22 human isolates were determined. stx_2d variants were most predominant among ovine isolates (141 of 146 [96.6%]). Of these, 119 were stx_2d-Ount positive, which was found in association with serotypes O75:H/H8/H40, O91:H, O123:H, O128:H2/H, OR:H2, and O153:H25/H. stx_2d-O111/OX3a subtypes were found in the remaining 22 ovine isolates, of serotypes O5:H, O91:H, O91:H2, and O123:H. The four ovine isolates of serotype O157:H/H21 possessed a stx_2vha subtype, and the single ovine O5:H isolate possessed a stx₂ subtype.

Of the 22 human STEC isolates, 10 possessed serotypes commonly associated with STEC derived from ovine feces (6a). Nine STEC isolates (six of serotype O128:H2, two of O91:H, and one of O123:H) were recovered from seven patients with diarrhea and from two asymptomatic carriers and possessed the stx_2d-Ount subtype. Furthermore, isolates OX3:H8 (Switzerland) and O5:H (Australia) were each recovered from HUS patients and possessed the stx_2d-Ount subtype and the stx_2d-OX3a and/or stx_2d-O111 subtypes, respectively. The O5:H isolate from the HUS patient is genetically indistinguishable from several epidemiologically unrelated O5:H isolates recovered from sheep by pulsed-field gel electrophoresis (31). Collectively, these observations suggest that this isolate had an ovine origin.

Twelve human isolates were of serotypes not commonly associated with sheep (O7:H, O8:H14, O15:H, O26:H, O91:H10, O91:H21, O121:H19, O145:H, and OX3:H8). These were recovered from patients with symptoms ranging from diarrhea to HUS and also included an isolate from a symptomless carrier. All of these isolates possessed stx₂ and stx_2vhb subtypes, and one isolate (O91:H21) contained two subtypes of stx₂ and stx_2vhb. However, it is important to emphasize that none of the human isolates from Switzerland possessed a serotype representative of the vast majority of isolates recovered from ovine sources. These data are consistent with studies by Pierard et al. (26) showing that STEC strains normally associated with human disease (serogroups O157, O111, O26, O103, and O145) do not possess a stx_2d subtype and that stx_2d-positive isolates are less frequently associated with HUS. These and previous studies reinforce the hypothesis that certain serotypes of STEC seem to be associated with their animal host species (4, 6a, 20). Studies in our laboratories demonstrate that STEC isolates recovered from ovine sources possess serotypes rarely observed among STEC isolates recovered from bovine sources (Hornitzky et al., unpublished results). Furthermore, we very rarely observe stx_2d subtypes among STEC isolates recovered from bovine sources in Australia (Brett et al., unpublished results). Collectively, these results are consistent with the observation that different stx₂ subtypes associate with certain serotypes and these data have significant ramifications in epidemiological studies of STEC infections. These observations also suggest that lamboid phages carrying different stx₂ subtypes lysogenize distinct E. coli populations, which may be determined by their serotype.

Vero cell assays of ovine isolates possessing stx_2d subtypes are generally toxigenic, with titers down to 10⁷ (6a). We did not determine the contribution of Stx1 toxin (which is present in almost all sheep isolates used in this study) to Vero cell toxicity. However, Paton et al. (24, 25) reported a low cytoxicity to Vero cells for the two stx_2d variants (stx_2d-O111 and stx_2d-OX3a), as did Pierard et al. (26) for the single isolate tested in that study. Pierard et al. (26) suggested that Stx2d-producing strains may be a marker for less-pathogenic STEC, since they often failed to possess associated virulence factors. We did not observe the eaeA gene among any of the ovine STEC isolates that possessed stx_2d in this study. This result is consistent with the observations of Pierard et al. (26), who failed to observe eaeA among 65 isolates displaying stx_2d variant genes. However, in contrast to the findings of Pierard et al. (26), 141 of 146 isolates recovered from ovine sources possessed the ehxA gene. These data suggest that further studies need to be carried out to determine the pathogenicity of ovine STEC to humans.

	ACKNOWLEDGMENTS

V.R. is a recipient of an Overseas Postgraduate Research Scholarship and a University of Wollongong Postgraduate Award. This work was supported by funds from Meat and Livestock, Australia.

We thank Jody Wilton and Wendy Forbes for technical assistance with sequencing and Kim Ziebel and Roger Johnson for serotyping the isolates from Switzerland.

	FOOTNOTES

^* Corresponding author. Mailing address: Elizabeth Macarthur Agricultural Institute, New South Wales Agriculture, Private Mail Bag 8, Camden, New South Wales 2570, Australia. Phone: 0061-246-406426. Fax: 0061-246-406384. E-mail: steve.djordjevic{at}agric.nsw.gov.au.

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Journal of Clinical Microbiology, May 2001, p. 1932-1937, Vol. 39, No. 5
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.5.1932-1937.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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