Use of Comparative Genomics As a Tool to Assess the Clinical and Public Health Signiﬁcance of Emerging Shiga Toxin-Producing Escherichia Coli Serotypes

MEAT SCIENCE Meat Science 71 (2005) 62–71 www.elsevier.com/locate/meatsci Review Use of comparative genomics as a tool to assess the clinical and public health signiﬁcance of emerging Shiga toxin-producing Escherichia coli serotypes

Mohamed A. Karmali *

Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, 110 Stone Road West, Guelph, Ont., Canada N1G 3W4

Abstract

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause sporadic or epidemic food- or water-borne illness whose clinical spectrum includes diarrhea, hemorrhagic colitis, and the potentially fatal hemolytic uremic syndrome (HUS). Over 200 STEC serotypes have now been implicated in human disease. Serotype O157:H7 is associated most outbreaks and most cases of HUS. Other serotypes are also associated with outbreaks and HUS but less commonly than serotype O157:H7, and some cause HUS but are typically non-epidemic. Many STEC serotypes have been associated with diarrhea, but not with outbreaks or HUS, while others, isolated from cattle, have never been linked to human disease. The only proven virulence strategies for STEC are Stx production and, in some strains, a characteristic attaching and effacing cytopathology on enterocyte that is mediated by factors encoded on a pathogenicity island (PAI) known as the locus of enterocyte effacement (LEE). But Stx subtypes and LEE cannot fully explain the apparent differences in virulence between STEC subgroups. However, publication of the genome sequences of two E. coli O157:H7 strains has revealed new candidate PAIs and has stimulated the use of novel approaches for assessing differences in virulence potential between groups of strains. This paper highlights the clinico-pathological features and pathogenesis of STEC infection, new infor- mation arising from E. coli O157:H7 genome sequences, and progress in the use of of comparative genomics for assessing potential differences in virulence and public health significance between STEC subgroups. Crown Copyright Ó 2005 Published by Elsevier Ltd. All rights reserved.

Keywords: Shiga toxin-producing Escherichia coli; Non-O157 STEC; Comparative genomics; Pathogenicity island; O-island 122; Public health

Contents

1. Introduction ...... 63 2. Clinicopathological features of STEC infection...... 63 3. Pathogenesis of STEC infection and virulence factors ...... 63 3.1. The attaching and effacing (AE) lesion and the locus of enterocyte effacement (LEE) ...... 64 3.2. Shiga toxins and the pathogenesis of HUS ...... 64 3.3. Plasmid-mediated virulence factors ...... 65 3.4. Other candidate virulence factors ...... 65 4. The genome of E. coli O157:H7 ...... 65 4.1. Pathogenicity islands (PAI) ...... 66 5. Comparative genomic approaches to assess the clinical and public health significance of STEC serotypes ...... 66

* Tel.: +1 519 822 3300. E-mail address: [email protected].

5.1. EDL 933 genomic O-island 122 (OI-122)...... 67 5.2. Incomplete OI-122...... 68 6. Conclusions...... 68 References ...... 68

1. Introduction 2001; World Health Organization, 1999). Others (e.g., O113:H21 and O91:H21) rarely cause outbreaks (Paton, Shiga toxin (Stx)-producing Escherichia coli (STEC) Woodrow, Doyle, Lanser, & Paton, 1999), but are asso- (Calderwood, Acheson, Goldberg, Boyko, & Don- ciated with sporadic episodes of HUS (Johnson et al., ohue-Rolfe, 1990), also referred to as Verocytotoxin- 1996; Nataro & Kaper, 1998). A large number of STEC producing E. coli (VTEC) (Konowalchuk, Speirs, & serotypes have been isolated from patients with diar- Stavric, 1977), are causes of a potentially fatal food- rhea, but have not been associated with outbreaks or or water-borne illness whose clinical spectrum includes HUS (Johnson et al., 1996; Nataro & Kaper, 1998; non-specific diarrhea, hemorrhagic colitis, and the World Health Organization, 1999), and yet others, iso- hemolytic uremic syndrome (HUS) (Karmali, 1989; lated from cattle, have never been linked to human dis- Karmali et al., 1985; Karmali, Petric, Steele, & Lim, ease (World Health Organization, 1999). Thus STEC 1983). The serious public health concern about STEC serotypes appear to differ in pathogenic potential, but infection is due to the risks of massive outbreaks (Carter the scientific basis for this is not known. Consequently et al., 1987; CCDR, 2000; CDC, 1993; Michino et al., the assessment of the clinical and public health risks is 1998; Riley et al., 1983) and of HUS, the leading cause greatly compromised when non-O157 STEC are found of acute renal failure in children (Karmali, 1989). Up in humans, foods, animals, and the environment. Publi- to 40% of the patients with HUS develop long-term re- cation of the genome sequences of two E. coli O157:H7 nal dysfunction and about 3–5% of patients die during strains (Hayashi et al., 2001; Perna et al., 2001)has the acute phase of the disease (Loirat et al., 1984; Siegler opened up new approaches for assessing differences in et al., 1991; Trompeter et al., 1983). There is no specific virulence potential between groups of strains. The objec- treatment for HUS, and vaccines to prevent the disease tive of this paper is to highlight the clinico-pathological are not yet available. features and pathogenesis of STEC infection, new infor- Ruminants, especially cattle, are the main reservoir of mation arising from whole genome sequencing of E. coli STEC, which are transmitted to humans primarily via O157:H7 strains, and progress in the use of of compar- contaminated foods and water (Griffin, 1995; Griffin & ative genomics for assessing potential differences in vir- Tauxe, 1991; Karch, Bielaszewska, Bitzan, & Schmidt, ulence between STEC subgroups. 1999; Karmali, 1989; Nataro & Kaper, 1998). Although serotype O157:H7 has been implicated in most outbreaks and in most cases of HUS globally (Griffin, 2. Clinicopathological features of STEC infection 1998; Karch et al., 1999; Karmali, 1989; Nataro & Ka- per, 1998), there is growing concern about the risk to hu- The characteristic features of STEC O157:H7 infec- man health associated with non-O157 STEC serotypes tion include a short period of abdominal cramps and (Johnson et al., 1996; Tarr & Neill, 1996), over 200 of non-bloody diarrhea, without significant fever, which which have now been associated with human illness may be followed, in some cases, by hemorrhagic colitis (World Health Organization, 1999). First reported in and the hemolytic uremic syndrome (HUS) (Karmali, association with HUS in Canada (Karmali et al., 1985; 1989). Karmali et al., 1983), non-O157 serotypes have since Defined by the triad of features: acute renal failure, been more commonly implicated in HUS than serotype thrombocytopenia, and microangiopathic hemolytic O157:H7 in Latin America (Lopez, Contrini, & de Rosa, anemia, HUS develops in about one-tenth to one quar- 1998) and Australia (Elliott et al., 2001a), and their fre- ter of the cases of STEC infection (Griffin, 1995; Griffin quency may be rising in Europe (Gerber, Karch, Aller- & Tauxe, 1991; Karch et al., 1999; Karmali, 1989; Nat- berger, Verweyen, & Zimmerhackl, 2002; Tozzi et al., aro & Kaper, 1998). 2003). Up to 20% of HUS cases in North America may be associated with non-O157 STEC (Banatvala et al., 1996). Some non-O157 STEC serotypes (e.g., sero- 3. Pathogenesis of STEC infection and virulence factors types O26:H11, O103:H2, O111:NM, O121:H19, and O145:NM) are associated with outbreaks and HUS The two main virulence strategies of STEC are Stx- but less commonly than serotype O157:H7 (Griffin & production and the ability to colonize the bowel (Nataro Tauxe, 1991; Johnson et al., 1996; McCarthy et al., & Kaper, 1998). E. coli O157:H7 and certain other 64 M.A. Karmali / Meat Science 71 (2005) 62–71

STEC serotypes (e.g., O26:H11, O103:H2, O111:NM, adhesin, intimin (Jerse et al., 1990; Nataro & Kaper, O121:H19, and O145:NM) colonize the mucosal epithe- 1998), and genes that encode the translocated intimin lial cells of the large bowel with a characteristic ‘‘attach- receptor known as Tir (Kenny et al., 1997) or EspE ing and effacing’’ (AE) cytopathology which is encoded (Deibel, Kramer, Chakraborty, & Ebel, 1998), and the for by genes on the locus of enterocyte effacement (LEE) Tir chaperone, CesT (Abe et al., 1999; Elliott et al., pathogenicity island (PAI) (Jerse, Yu, Tall, & Kaper, 1999). The LEE 4 operon encodes the structural needle 1990; McDaniel & Kaper, 1997). Serotypes associated protein, EscF (Wilson, Shaw, Daniell, Knutton, & Fran- with the AE cytopathology are also referred to as kel, 2001), the translocator proteins EspA, EspB, and enterohemorrhagic E. coli (EHEC) (Levine et al., EspD (Nataro & Kaper, 1998), and the effector protein 1987). While Stxs and LEE-encoded factors are the only EspF (McNamara & Donnenberg, 1998). Other LEE- proven virulence components, STEC strains produce encoded effector proteins include EspG (Elliott et al., many other putative virulence factors whose precise role 2001b), EspH (Tu, Nisan, Yona, Hanski, & Rosenshine, in pathogenesis is not known. E. coli O157:H7 has a low 2003), and Map (Kenny & Jepson, 2000). The TTSS also infectious dose (100–500 organisms) (Griffin, 1998), secretes a number of other effector molecules that are en- which is thought to reflect its resistance to gastric acid coded outside the LEE on other pathogenicity islands. (Lin et al., 1996; Waterman & Small, 1996). After inges- The precise role of these so-called non-LEE-encoded tion, the organism passes through the stomach and effectors (NLEs) (Deng et al., 2004; Gruenheid et al., small bowel and is thought to colonize enterocytes of 2004) has yet to be fully established. The first gene the large bowel mucosa by a characteristic attaching (Ler), in the LEE 1 operon, activates transcription of and effacing (AE) cytopathology (Nataro & Kaper, the LEE 2, 3, and 4 operons (Elliott et al., 2000; Mellies, 1998). Elliott, Sperandio, Donnenberg, & Kaper, 1999), which, in E. coli O157:H7, are also activated by quorum sensing 3.1. The attaching and effacing (AE) lesion and the locus through activation of autoinducer-2 (Sperandio, Mel- of enterocyte effacement (LEE) lies, Nguyen, Shin, & Kaper, 1999).

The AE lesion of E. coli O157:H7 and other EHEC 3.2. Shiga toxins and the pathogenesis of HUS serotypes resembles that associated with enteropathogenic E. coli (EPEC) and consists of the destruction of Human STEC strains elaborate at least four potent microvilli, an intimate effacing adherence of the organ- bacteriophage-mediated Shiga toxins (Melton-Celsa & ism to the enterocyte membrane, and changes in the OÕBrien, 1998; Nataro & Kaper, 1998): Stx 1, Stx 2, cytoskeletal structure of the enterocyte associated with Stx 2c, and Stx 2d. Each may be present alone, or in a the accumulation of polymerized actin and other cyto- combination of two or three different StxÕs. Stx1 is virtu- skeletal proteins beneath the site of bacterial attachment ally identical to Shiga toxin, but is serologically distinct (Knutton, Lloyd, & McNeish, 1987; Moon, Whipp, from Stx 2s (Karmali, 1989; OÕBrien et al., 1992). The Arzenio, Levine, & Gianella, 1983; Nataro & Kaper, toxins share a common polypeptide subunit structure 1998; Sherman, Soni, & Karmali, 1988). All the viru- consisting of an enzymatically-active A subunit lence factors necessary for the formation of the AE le- (32 kDa) linked to a pentamer of B subunits sion, in EHEC and EPEC, are encoded by the LEE (7.5 kDa). StxÕs are produced in the bowel and are PAI which encodes the structural, accessory, and effec- translocated intact into the circulation although the tor molecules of a type III secretion system (TTSS) mechanism of translocation is not well understood. (Foubister, Rosenshine, Donnenberg, & Finlay, 1994; The toxins are thought to be transported by leukocytes Goosney, DeVinney, & Finlay, 2001; Jarvis et al., (te Loo et al., 2000) to capillary endothelial cells in the 1995; Knutton et al., 1994; Rosenshine, Donnenberg, renal glomeruli, gastrointestinal tract, pancreas, and Kaper, & Finlay, 1992), a macromolecular complex other organs and tissues (Monnens, Savage, & Taylor, spanning both bacterial membranes that is used by 1998; Richardson et al., 1992). After binding to the gly- many Gram-negative bacterial pathogens to inject viru- colipid receptor, globotriaosylceramide (Gb3) (Ling- lence factors directly into host cells to subvert host cel- wood et al., 1987), on the target endothelial cell, the lular function for the benefit of the pathogen (Hueck, toxins are internalized by receptor-mediated endocytosis 1998). The LEE of STEC serotype O157:H7 reference (Sandvig, Olsnes, Brown, Peterson, & v. Deurs, 1989). strain EDL 933, which is 43.4 kb in size, contains 41 They then target the endoplasmic reticulum via the open reading frames (ORFs) which are organized in five Golgi by a process termed ‘‘retrograde transport’’ polycistronic operons (LEE 1, LEE 2, LEE 3, LEE 5, (Sandvig et al., 1993). Inside the host cell, the A subunit and LEE 4) (Nataro & Kaper, 1998). LEE 1, LEE 2, is proteolytically nicked to give an enzymatically active and LEE 3 encode structural and regulatory compo- A1 fragment (OÕBrien et al., 1992), which cleaves the nents of the TTSS (Nataro & Kaper, 1998). LEE 5 con- N-glycosidic bond at position A4324 of the 28S rRNA tains the eae gene, which encodes the outer membrane of the 60S ribosomal subunit (Endo et al., 1988). This M.A. Karmali / Meat Science 71 (2005) 62–71 65 blocks EF 1-dependent aminoacyl tRNA binding, to be involved in the efficient colonization of cattle (Ste- resulting in the inhibition of protein synthesis (OÕBrien vens, van Diemen, Frankel, Phillips, & Wallis, 2002), et al., 1992). Stxs may also damage eukaryotic cells by and Iha (Tarr et al., 2000), a putative chromosomally- apoptosis (Monnens et al., 1998). Cytokines, especially encoded adhesin in E. coli O157:H7, that is similar to TNF-a and IL 1-b, potentiate toxin action through the iron regulated gene A (irgA) of Vibrio cholerae. Like upregulation of the cellular receptor; Gb3 (Monnens et many other enteric pathogens, E. coli O157:H7 is able to al., 1998). It is thought that increased cytokine produc- utilize heme and/or hemoglobin as iron sources (Law & tion might be the result of Stx action on monocytes (van Kelly, 1995). ChuA, an outer membrane protein, is Setten, Monnens, Verstraten, van den Heuvel, & van thought to be a specific receptor for heme uptake in this Hinsbergh, 1996). organism (Mills & Payne, 1995). Knowledge about the While the injurious action of Stxs on endothelial cells intestinal colonization mechanisms of LEE-negative appears to be crucial to the development of HUS, the STEC is limited. The LEE-negative serotype O113: precise cellular events that result in the associated path- H21 strain, CL-15 was shown to adheres to HEp2 cells ophysiological changes, including thrombotic microan- in vitro and to colonizes the rabbit intestine, and was giopathy, hemolytic anemia and thrombocytopenia, associated with microvillus effacement, but without evi- remain to be elucidated. dence of actin condensation at the site of attachment The LEE and the Shiga toxins are the only virulence (Dytoc et al., 1994). The precise mechanism of adher- factors proven to have a role in the genesis of STEC- ence is unknown. Doughty et al. (2002) using wild-type associated disease. The contributions of various host and mutant strains, showed that long polar fimbria (age, immunity, receptor type and distribution, inflam- (Lpf) genes are involved in the adherence of O113:H21 matory response, and genetic factors) and parasite strains to epithelial cells in vitro. Several of these puta- determinants (infectious dose, toxin types, and other tive virulence genes including efa1, iha, and lpf, are chromosomal or plasmid-encoded virulence factors) to now known to be part of newly-described pathogenicity disease susceptibility and severity remain to be fully islands (PAIs) in the E. coli O157:H7 genome. understood. Some other plasmid- or chromosomal factors that have been postulated, but not proved, to be involved in the pathogenesis of STEC infection are 4. The genome of E. coli O157:H7 discussed below. Factors that apparently make STEC serotype 3.3. Plasmid-mediated virulence factors O157:H7 more virulent than other serotypes or that con- fer different degrees of virulence to different serotypes E. coli O157:H7 and other EHEC contain a large 92- and strains are poorly understood. However the publi- kb F-like plasmid, pO157, which contains 100 ORFs of cation of the whole genome sequence of two epidemic which 20 encode putative virulence factors (Burland et strains of E. coli O157:H7, EDL 933 in the United States al., 1998; Karch, Schmidt, & Brunder, 1998; Makino (Perna et al., 2001), and the Sakai strain in Japan (Hay- et al., 1998). These include: the EHEC hemolysin (hly- ashi et al., 2001), has greatly accelerated research to re- CABD operon); catalase-peroxidase (katP); a serine solve these knowledge gaps. protease (espP) which is secreted by a type IV secretion Both genomes (Hayashi et al., 2001; Perna et al., system; a 13 gene cluster (etpC–etpO) related to the type 2001) have a size of 5.5 Mb, compared to the 4.6-Mb II secretion pathway; and an ORF encoding a 3169 aa genome size of E. coli K-12 strain MG1655. The O157 predicted product that shares homology with large clos- and K-12 genomes share a common backbone of tridial toxins. None of these genes has been shown to 4.1 Mb, which is co-linear except for one 422-kb inver- have a role in pathogenesis. The large plasmid is present sion spanning the replication terminus in EDL 933, in several other EHEC serotypes, but its composition is but not in the Sakai genome. K-islands (KI), reflect dif- very heterogeneous (Brunder, Schmidt, Frosch, & ferences between the EDL 933 and MG 1655 genomes, Karch, 1999). Plasmids from strains causing HUS have which are DNA segments present in MG 1655, but lacked one or more of hly, katP, and espP, suggesting not in EDL933, and O-islands (OI), which are unique that the latter are not, by themselves, critical for the segments in EDL933 (Perna et al., 2001). OIs total development of severe disease (Bielaszewska et al., 1.34 Mb of DNA, and KIs total 0.53 Mb. There are 2000). 177 OIs and 234 KIs greater than 50 bp. Overall, EDL933 contains 1387 new genes, encoded in strain- 3.4. Other candidate virulence factors specific clusters of diverse sizes. Several OIs (e.g., OIs # 1, 7, 28, 43, 47, 48, 115, 122, 138, 141, and 154) encode These include Efa1 (Nicholls, Grant, & Robins- putative virulence factors that may be of relevance to the Brown, 2001), a chromosomally-encoded adhesin, iden- pathogenesis of STEC. Eighteen multigenic regions are tified in serotype O111:NM STEC strain, that is thought related to known bacteriophages, including the stx-2 66 M.A. Karmali / Meat Science 71 (2005) 62–71 converting phage BP-933W and the stx-1 converting as those newly identified in the E. coli O157:H7 genome) phage CP-933V (Perna et al., 2001). may also be critical for the virulence of STEC strains.

4.1. Pathogenicity islands (PAI) 5. Comparative genomic approaches to assess the clinical and public health significance of STEC serotypes The term PAI refers to a large tract of genomic DNA, containing several virulence genes, that is absent from To investigate the basis for the apparent differences in non-pathogenic members, or less pathogenic members, virulence between STEC serotypes and strains we have of a species (Hacker & Kaper, 2001). Typically, PAIs classified STEC serotypes into five ‘‘seropathotypes’’ are inserted into tRNA genes, are often flanked by direct (A–E) based on the relative frequencies of these sero- repeat sequences, contain mobile genetic elements, and types in human disease and on their association with have a significantly different G + C content and codon outbreaks and HUS relative to serotype O157:H7 (Kar- usage patterns than the host genome, suggesting they mali et al., 2003; Table 1). were acquired by horizontal transfer (Hacker & Kaper, Seropathotype-A comprises serotypes O157:H7 and 2001). O157:NM, which are the most common causes of out- Increasing evidence suggests that major differences in breaks and HUS. Seropathotype-B includes serotypes virulence between groups of strains in species such as E. O26:H11, O103:H2, O111:NM, O121:H19, and coli, Salmonella spp. and Helicobacter pylori are related O145:NM that are associated with outbreaks and to the presence of PAIs (Hacker et al., 2001; Kaper & HUS, but less commonly than serotype O157:H7. Sero- Hacker, 1999). For example, the LEE PAI appears to pathotype-C consists of serotypes O91:H21, O113:H21, correlate with virulence in STEC, since virtually all ser- and many others that are associated with sporadic otypes that are associated with outbreaks and HUS are HUS but not epidemics. Seropathotype-D contains LEE-positive. On the other hand, serotype O157:H7 is many serotypes that are associated with diarrhea but associated with outbreaks and HUS much more com- not with outbreaks or HUS, and Seropathotype-E commonly than other LEE-positive serotypes such as prises multiple animal STEC serotypes that have never O26:H11, O103:H2, O111:NM, O121:H19, and been associated with human disease. O145:NM, and some LEE-negative serotypes are also The seropathotype classification (Karmali et al., associated with HUS (Nataro & Kaper, 1998), and 2003) provides a framework for comparing strains of rarely with outbreaks (Paton et al., 1999). Other LEE- differing pathogenic potential using several advanced positive serotypes from bovines have never been associ- techniques such as comparative genomics, proteomics, ated with human disease (World Health Organization, and transcriptomics. This can be a powerful approach 1999). The scientific basis for these apparent differences to identify the critical differences in virulence capability, in virulence between STEC subgroups is thus not fully including PAIs, that enables some seropathotypes to understood. It is likely that, in addition to LEE, other cause epidemic and more severe disease than others. hitherto unknown factors, possibly other PAIs (such Once candidate PAIs or other genetic elements are iden-

Table 1 Classification of STEC serotypes into seropathotypes (Karmali et al., 2003) Seropathotype Relative incidence Outbreaks Severe disease (e.g., HUS) Serotypes (examples) A High Common Yes O157:H7 O157:NM B Moderate Uncommon Yes O26:H11 O103:H2 O111:NM O111:H8 O121:H19 O145:NM C Low Rare Yes O91:H21 O104:H21 O113:H21 Many others D Low Rare No Multiple E Non-human Not applicable Not applicable Multiple M.A. Karmali / Meat Science 71 (2005) 62–71 67 tified, definitive evidence for virulence would require progressive decrease in the frequency of complete OI- demonstration of pathogenicity in experimental in vitro 122 (COI-122) from seropathotypes A to E as shown and in vivo models using wild type and mutant strains. in Fig. 1. The difference in frequency of COI#122 between 5.1. EDL 933 genomic O-island 122 (OI-122) seropathotypes A, B, and C (associated with HUS) and D and E (not associated with HUS) was highly sig- Following promising preliminary screening studies nificant (p < 0.0001), as was the difference in frequency we investigated the seropathotype distribution of OI- of OI#122 between seropathotypes A and B (associated 122 which is a 23,029 base pair genomic island (Perna with epidemic disease) and C, D, and E (not associated et al., 2001) that is composed of 26 open reading frames with epidemic disease) (Karmali et al., 2003). including those that show significant homology to viru- The study also confirmed a close relationship between lence genes namely Salmonella typhimurium pagC(Pul- eae (a stable marker of LEE) and seropathotypes that kkinen & Miller, 1991), Shigella flexneri enterotoxin 2 are associated with serious and/or epidemic disease (sen)(Nataro et al., 1995), and the EHEC factor for (Fig. 2)(Karmali et al., 2003). adherence (efa1) (Nicholls et al., 2001) which is also re- It was noteworthy (Karmali et al., 2003), that strains ferred to as lymphocyte inhibition factor (lifA) (Klapp- that were COI-positive were always LEE-positive roth et al., 2000). OI#122 has several features that are whereas there were many LEE-positive strains that were consistent with the properties of a PAI. It is adjacent COI-negative. Furthermore, in serotypes that were rep- to a pheV tRNA locus. The terminus closest to the pheV resented by more than one strain, all strains in the same locus consists of a P4 integrase gene and four sequences serotype gave an identical pattern for OI#122 (either that show homology to ISEc8. The downstream termi- ‘‘complete’’, ‘‘incomplete’’ or ‘‘absent’’) and for eae. nal region consists of six sequences homologous to This supports our approach of postulating virulence dif- ISEc8 and IS629. OI#122 also contains two putative ferences between seropathotypes based on the presence transposases and nine genes of unknown function. or absence of specific PAIs rather than on the basis of Two of these genes of unknown function have recently been shown to be non-LEE-encoded effector molecules (Deng et al., 2004). The study strains for our investigation (Karmali 100 et al., 2003) are shown in Table 2. 80 All strains were tested for the following four EDL 60 933 OI#122 virulence genes by PCR (Karmali et al., Complete %+ve 40 2003): Z4321 (pagC homolog), Z4326 (sen homolog), Incomplete Z4332 (efa1 homolog), and Z4333 (efa1 homolog). Neg- 20 Absent ative PCR reactions were confirmed by Southern hybridization. 0 ABCDE Overall 28 (40%) strains contained OI#122 (positive Seropathotype for all four virulence genes), 27 (38.6%) contained an ‘‘incomplete’’ OI#122 (positive for 1–3 genes), and 15 Fig. 1. Seropathotype distribution of complete and incomplete OI# (21.4%) strains did not contain OI#122. There was a 122 (Karmali et al., 2003).

Table 2 Strains and serotypes used to study the seropathotype distribution of 100 OI#122 (Karmali et al., 2003) 90 Seropathotype Number Serotypes 80 of strains 70 A 13 O157:H7 (10 strains); O157:NM (3 strains) 60 B 15 O26:H11 (3); O103:H2 (3); O111:NM (3); 50 O121:H19 (3); O145:NM (3) %+ve 40 C 14 O5:NM (2); O91:H21 (4); O104:H21 (1); O113:H21 (4); O121:NM (2); O165:H25 (1) 30 D 14 O7:H4 (1), O69:H11 (1); O103:H25 (2); 20 O113:H4 (1), O117:H7 (2);O119:H25 (1); 10 O132:NM (1); O146:H21 (1), O171:H2 (1), 0 O172:NM (1), O174:H8 (1), OR:H2 (1) ABCDE E 14 O6:H34 (1); O8:H19 (1); O39:H49 (1); Seropathotype O46:H38 (1); O76:H7 (1); O84:NM (1); O88:H25 (1); O98:H25 (1); O113:NM (1); COI-122 LEE O136:NM (1); O136:H12 (1); O153:H31 (1); O156:NM (1); O163:NM (1) Fig. 2. Seropathotype distribution of COI-122 and LEE. 68 M.A. Karmali / Meat Science 71 (2005) 62–71 individual virulence genes. In contrast to OI#122 and syndrome: a family outbreak. Journal of Pediatric Infectious eae, Stx genes and putative plasmid-encoded putative Diseases, 15, 1008–1011. virulence genes (hlyA, espP, and katP) showed variabil- Bielaszewska, M., Schmidt, H., Liesegang, A., Prager, R., Rabsch, W., Tschape, H., et al. (2000). Cattle can Be a reservoir of sorbitol- ity among individual strains belonging to the same sero- fermenting Shiga toxin-producing Escherichia coli O157:H(À) type in several instances indicating that these genes are strains and a source of human diseases. Journal of Clinical not suitable for exploring virulence differences between Microbiology, 38, 3470–3473. seropathotypes. Brunder, W., Schmidt, H., Frosch, M., & Karch, H. (1999). The large plasmids of Shiga-toxin-producing Escherichia coli (STEC) are highly variable genetic elements. Microbiology, 145(5), 1005–1014. 5.2. Incomplete OI-122 Burland, V., Shao, Y., Perna, N. T., Plunkett, G., Sofia, H. J., & Blattner, F. R. (1998). The complete DNA sequence and analysis of The significance of the incomplete OI-122 ( Fig. 1)is the large virulence plasmid of Escherichia coli O157:H7. Nucleic not fully understood. One third of the 27 strains with Acids Resources, 26, 4196–4204. ‘‘incomplete’’ OI#122 were lacking only one of the four Calderwood, S. B., Acheson, D. W. K., Goldberg, M. B., Boyko, S. A., & Donohue-Rolfe, A. (1990). A system for production and rapid OI#122 genes tested (in most cases pagC) whereas two- purification of large amounts of the Shiga toxin/Shiga-like toxin I B thirds were negative for two or three of the four genes subunit. Infection and Immunity, 58, 2977–2982. (Karmali et al., 2003). Most of the strains (6 of 9) lack- Carter, A. O., Borczyk, A. A., Carlson, J. A. K., Harvey, B., Hockin, ing only one gene (pagC) were in seropathotype B. It is J. C., Karmali, M. A., et al. (1987). A severe outbreak of likely that many strains in this category are part of a Escherichia coli O157:H7-associated hemorrhagic colitis in a nursing home. New England Journal of Medicine, 317, 1496–1500. mosaic pathogenicity island (Morabito, Tozzoli, Os- CCDR (2000). Waterborne outbreak of gastroenteritis associated with wald, & Caprioli, 2003; Tauschek, Strugnell, & Rob- a contaminated municipal water supply, Walkerton, Ontario, ins-Brown, 2002; Zhu et al., 2001) comprising LEE May–June 2000. Canada Communicable Disease Report, 26, and OI-122 (without the pagC gene) in which one end 170–173. of OI-122 is integrated into LEE. On the other hand CDC (1993). Update: multistate outbreak of Escherichia coli O157:H7 infections from Hamburgers – Western United States, 1992–1993. all 18 strains that lacked two or three genes (Karmali, Journal of American Medical Association, 269, 2194–2196. 1992) were distributed in seropathotypes C, D, and E. Deibel, C., Kramer, S., Chakraborty, T., & Ebel, F. 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