INFECTIONS WITH VEROTOXIN-PRODUCING ESCHERICHIA COLI ...

-(CME 3182) Infections with VTEC O157:H7, O104:H4 and others

Continuing Medical Education

INFECTIONS WITH VEROTOXIN-PRODUCING ESCHERICHIA COLI O157:H7 AND OTHER SEROTYPES, INCLUDING THE OUTBREAK STRAIN O104:H4 Buvens G, Piérard D Belgian Reference Laboratory for VTEC/STEC, Department of Microbiology and Infection Control, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels, Belgium Correspondence and offprint requests to: Glenn Buvens, E-mail: [email protected]

ABSTRACT Through the acquisition of mobile genetic elements, the normally harmless commensal Escherichia coli evolved into a highly adapted human pathogen. Pathogenic strains of E. coli are associated with urinary tract infections, sepsis/meningitis, and diarrhoea. At least six different diarrhoeagenic E. coli pathotypes have emerged during the past three decades as human pathogens of public health importance worldwide. In this review, we focus on the clinical features, pathogenic mechanisms, and diagnostic strategies of verotoxin-producing E. coli (VTEC) that are associated with sporadic cases and epidemics of gastrointestinal disease throughout the world. Recently, an E. coli strain of serotype O104:H4 combining verotoxin production with virulence factors of another pathotype, the enteroaggregative E. coli (EAEC), emerged as the cause of a severe outbreak in Europe. Key words: Diarrhoeagenic E. coli, verotoxin-producing and enteroaggregative E. coli

INTRODUCTION Escherichia coli are the most abundant Gram-negative facultative anaerobes of the colonic flora in mammals and birds where they live as commensals with mutual benefit for bacteria and host. E. coli break down cellulose, assist in the absorption of vitamin K by the host, and prevent the colonization of the intestinal mucosa by diarrhoeagenic bacteria (1). While in most cases E. coli is a harmless inhabitant of the gut,

doi: 10.2143/ACB.67.0.2062000

“non-pathogenic” strains may cause disease in immunosuppressed hosts or when gastrointestinal barriers are violated. Moreover, through the acquisition of several virulence factors, such as adhesins, invasins, cyto- and enterotoxins encoded on mobile genetic elements (chromosomal pathogenicity islands, extra-chromosomal plasmids, bacteriophages, and transposons) several pathogenic E. coli types (pathotypes) have emerged during recent decades. Infections with pathogenic E. coli strains are associated with three clinical syndromes: 1) urinary tract infection, 2) sepsis/meningitis, and 3) gastroenteritis (1). This review will focus on diarrhoeagenic E. coli, in particular verotoxin-producing E. coli (VTEC) with special emphasis on verotoxin producing/enteroaggregative E. coli O104:H4 (VT/EAEC), that recently caused a severe outbreak in Europe.

PATHOTYPES OF DIARRHOEAGENIC ESCHERICHIA COLI Diarrhoeagenic E. coli (DEC) are increasingly recognized as an underestimated cause of gastrointestinal disease worldwide. As presented in Table 1, six pathotypes of DEC were described: enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), verotoxin- or Shiga toxin-producing E. coli (VTEC or STEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusely adherent E. coli (DAEC) (2). Each pathotype exhibits specific characteristics related to epidemiology, pathogenesis, clinical manifestations, and treatment. The strategy of infection followed by DEC includes four steps: 1) mucosal colonization, 2) evasion of host defenses, 3) multiplication, and 4) host damage (3). The first step of adhesion to the intestinal mucosa is the most conserved feature among diarrhoeagenic E. coli. The next steps in infection, however, show remarkable variety.

Acta Clinica Belgica, 2012; 67-?

1

2

Infections with VTEC O157:H7, O104:H4 and others

Research and diagnostics have primarily focused on infections by VTEC because of their worldwide potential to cause widespread foodborne outbreaks of bloody diarrhoea, often complicated by the haemolytic uraemic syndrome (HUS). Other DEC pathotypes have received little attention due to the lower severity of the caused disease and to the assumed lower incidence in industrialized countries. In particular, EAEC cause less severe, self-limiting gastrointestinal disease, and are thought to occur predominantly in developing countries. The recent German outbreak with a hybrid E. coli O104:H4 exhibiting characteristics of both EAEC and VTEC, however, highlights the importance of a better understanding of the epidemiology and pathogenicity of both pathotypes. Enteropathogenic E. coli (EPEC) are linked to outbreaks and sporadic cases of infantile diarrhoea in the developing world which can be severe and prolonged. Characteristic for EPEC infections is the attaching and effacing (A/E) histopathology which encompasses effacement of the intestinal microvillous brush border and intimate attachment of the bacterium to the epithelial cell. A/E lesions are marked by an actin polymerization forming a pedestal structure directly beneath the adhering bacterium. Three steps were identified in EPEC pathogenesis: 1) localized adherence through bundle-forming pili (BFP) encoded by the EPEC adherence factor (EAF) plasmid, 2) signal transduction activity encoded on the locus of enterocyte effacement (LEE) pathogenicity island, encoding a type III secretion system, secretory proteins (espA, espB, and espD), and the bacterial adhesin intimin (eaeA), and 3) intimate adherence and pedestal formation. EPEC can be divided into “typical” and “atypical” EPEC, depending on their ability to form BFPs. Symptomatic and asymptomatic children and asymptomatic adults are the main reservoir of EPEC, which are transmitted through faecal-oral contamination. Enterotoxigenic E. coli (ETEC) are associated with traveller’s diarrhoea, watery diarrhoea in young children in developing countries, and diarrhoea in piglets. After ingestion, ETEC adhere to small bowel enterocytes by peritrichous fimbriae or colonization factor antigens (CFAs). They produce one or two plasmid-encoded enterotoxins, designated heatlabile (LT) and heat-stabile (ST) toxins. LT and ST cause inhibition of sodium absorption and stimulation of chloride secretion, which leads to watery diarrhoea. Humans are the main reservoir of ETEC, and faecal contamination of food and water is the most frequent mode of transmission. Verotoxin-producing E. coli (VTEC) are associated with sporadic and epidemic cases of watery and bloody diarrhoea, which may progress to haemorrhagic colitis and HUS. The lifethreatening HUS is characterized by acute renal failure, haemolytic anaemia, and thrombocytopenia. It affects especially young children and the elderly, but this complication can be observed in all age groups. The cardinal virulence trait of VTEC leading to HUS is its ability to produce cytotoxins called vero(cyto)toxins (VT) or Shiga toxins (Stx). Although the diarrhoea caused by these micro-organisms is often not bloody, a third denomination referring to this clinical characteristic has been proposed for the most pathogenic strains, for instance the well-known serotype O157:H7, enterohaemorrhagic E. coli (EHEC). However, since the essential virulence features defining organisms able to cause HUS, haemorrhagic colitis and bloody diarrhoea are not clear, this denomination is confusing, being used with variable definitions by the authors.


Enteroaggregative E. coli (EAEC) have been implicated in endemic diarrhoea of infants in both industrialized and developing countries, persistent diarrhoea among HIV patients and traveller’s diarrhoea. EAEC show an aggregative adherence (AA) to HEp-2 cells in a distinct “stacked-brick” pattern. The AA phenotype is encoded by bundle-forming pili termed aggregative adherence fimbriae (AAF) located on a 55-65-MDa plasmid (pAA). EAEC pathogenesis comprises three major components: 1) aggregative adherence to the intestinal mucosa, 2) production of entero- and cytotoxins such as the heat-stabile enterotoxin EAST, and 3) induction of mucosal inflammation. Several outbreaks of EAEC have been described worldwide, but no natural reservoir has been identified. Enteroinvasive E. coli (EIEC) are closely related to Shigella spp. Both organisms have the ability to invade colonic epithelial cells mediated by a series of plasmid and chromosomal loci. EIEC infection usually presents as watery diarrhoea similar to the secretory diarrhoea seen with ETEC. Only a minority of patients experience the dysentery syndrome associated with Shigella spp. Humans are the main reservoir of EIEC, which are transmitted via faecal-oral contamination of food and water. Diffusely adherent E. coli (DAEC) express a characteristic, diffuse adherence pattern to Hep-2 cells, distinct from the localized adherence of EPEC and VTEC and the aggregative adherence of EAEC. A fimbrial adhesin called F1845 has been observed in approximately 75% of DAEC strains around the world. Adherence to HEp-2 cells induces the development of “finger-like” cellular extensions that wrap around the adherent bacteria. Although DAEC have been incriminated as a cause of watery diarrhoea in young children between one and five years of age, the role of this phenotype in pathogenesis is not yet understood.

VEROTOXIN-PRODUCING ESCHERICHIA COLI OF SEROTYPE O157:H7 AND OTHERS History

Since 1983, VTEC, also known as STEC, have been associated with foodborne diarrhoea (often bloody), haemorrhagic colitis, and HUS. VTEC were first described in 1977 by Konowalchuk et al. when culture filtrates from some E. coli strains produced an irreversible cytotoxic effect in Vero cells (4). Later, O’Brien reported that extracts of certain E. coli strains were cytotoxic for HeLa cells, and that this activity could be neutralized by antiserum against Shigella dysenteriae type 1 (Shiga) toxin (Stx) (5). It was subsequently shown that the Verotoxin and the Shiga toxin are structurally and antigenically similar. The clinical importance of VTEC was first recognized by Riley et al. in 1982 when VTEC of serotype O157:H7 were isolated from patients associated with two outbreaks of haemorrhagic colitis following the consumption of undercooked hamburgers (6). Also in 1983, Karmali et al. reported the association of sporadic cases of HUS with VTEC O157:H7 infection (7).

Clinical features

VTEC have been associated with sporadic and epidemic cases of watery or bloody diarrhoea worldwide. While over 380 different VTEC serotypes have been described, only a few serotypes have been frequently associated with severe human disease, such as haemorrhagic colitis and HUS, especially in young


children and the elderly (8). Infection with some serotypes, mainly O157:H7/H-, O26:H11/H-, O103:H2/H-, O111:H8/H-, and O145:H28/H-, is clearly associated with a higher risk to develop these complications and are often referred to as enterohaemorrhagic E. coli (EHEC). However, the essential virulence and genetic factors associated with the development of haemorrhagic colitis and HUS are poorly understood and we prefer not to refer to this denomination, but rather to the seropathotype classification proposed by Karmali et al. (9, 10), as presented in Table 2. Most of our knowledge about VTEC pathogenesis originates from work done on VTEC O157:H7, which is considered the most clinically significant serotype. HUS is the most serious complication associated with VTEC infection, and is characterized by microangiopathic haemolytic anaemia, thrombocytopenia, and acute renal failure, often preceded by an episode of bloody diarrhoea (11). HUS is the most important cause of acute nephropathy in young children worldwide (12), causing death in about 5 to 10% of affected patients. Moreover, 10-20% of the survivors present renal or non-renal sequelae, such as hypertension and central nervous system manifestations. A multi-centre study done by Piérard et al. in 1996 revealed an association between HUS and VTEC infection in at least 60% of all HUS patients in Belgium (13). The incidence of HUS was 4.3 cases/100,000 children under 5 years of age, and 0.42/100,000 when patients of all ages were taken into account.

circulation. The main target cells are microvascular endothelial cells expressing Gb3 and Gb4 in kidney, brain, and gastrointestinal mucosa. The cell-specific expression of Gb receptors might underlie the organ-specific manifestations of HUS. The endothelial injury elicited by VT result in coagulation activation, leading to accumulation of platelet-fibrin thrombi causing ischemic damage to colon, kidneys and other tissues. There is also evidence that at the cellular level proinflammatory cytokines and chemokines act synergistically with verotoxins to injure host cells (11). Additional virulence factors are the genomic pathogenicity island LEE, which is shared by VTEC and EPEC, and an enterohemolysin. LEE encodes the structural components of a type III secretion system (TTSS), an outer membrane protein called intimin (Eae) and its receptor, the translocated intimin receptor (Tir) (19). The TTSS functions as a molecular syringe whereby effector molecules are translocated directly into the host cell interfering with its cytoskeleton and other cellular processes (20). The enterohemolysin produced by most pathogenic VTEC strains is cytolytic to human endothelial cells (21). This pore-forming hemolysin probably induces the release of hemoglobin from red blood cells, thereby providing a source of iron that may stimulate the growth of VTEC in the gut.

Treatment

VTEC are emerging pathogens: they were first discovered in 1983 after two small outbreaks of haemorrhagic colitis (6). While only rare isolates of serotype O157:H7 could be found in previous reports in subsequent years many outbreaks were reported (22). Overall, sporadic cases are however more frequent than outbreak-related cases. In Belgium, VTEC represent the third bacterial pathogen in stools, with one fifth of isolates belonging to serogroup O157 (23). However, it represent the majority of documented infections in HUS cases (13). Like in other countries of continental Europe before the large outbreak observed this year, only a few small-scale outbreaks were reported (24).

The treatment of VTEC infections and of HUS is supportive. Antibiotics could hasten the occurrence of HUS, through increased production and release of VT. However, this in vitro observation has not been formally confirmed in clinical studies and this point remains controversial (14). Since they do not shorten the duration of symptoms, antibiotics are considered to be contra-indicated in the treatment of VTEC.

Pathogenesis and virulence genes

VTEC pathogenicity is determined by the production of multiple virulence factors of which VT are considered the most important. Two major types, VT1 and VT2, have been identified and show distinct immunogenic and genetic properties. Multiple subtypes of VT1 (VT1a, VT1c, and VT1d) and VT2 (VT2a to VT2g) have been described with significant differences in biologic activity, serologic reactivity and receptor binding (15). Many isolates produce several types or subtypes of VT. VTEC strains that produce VT2a, VT2c, and/or the elastase-activatable VT2d seem to be more often associated with HUS and bloody diarrhoea, while strains producing VT1, VT2b, or VT2e are associated with milder or asymptomatic infections (16, 17, 18). VT are AB5 toxins comprising one A subunit exhibiting an N-glycosidase enzymatic activity and a B pentamer that is responsible for binding to the eukaryotic glycolipid receptors globotriaosylceramide (Gb3) or globotetraosylceramide (Gb4). After binding to its receptor, the entire holotoxin-receptor complex is internalized by receptormediated endocytosis. The N-glycosidase activity of the A peptide causes depurination of a critical residue in the 28S rRNA of 60S ribosomes resulting in protein synthesis inhibition. The genes encoding for these toxins are carried by bacteriophages and can be rapidly gained and lost by horizontal transfer. VT are produced in vivo in the intestines after colonization by VTEC and then cross the intestinal wall into

Epidemiology

Source of infection

VTEC are present in the intestine of many animals. Ruminants are frequently colonized without disease symptoms by VTEC O157, the serogroup most frequently associated with outbreaks and severe infection. In a recent Belgian study at farm level, it was shown that the overall farm prevalence of VTEC O157 was 37.8%, with a prevalence in individual cattle varying from 0 to 85% (25). The most frequent vehicle of infection identified in outbreaks is meat, in particular insufficiently cooked ground beef: in the process of mincing, the surface contamination is brought to the internal parts of the meat, where it escapes to sufficient temperatures to destroy the micro-organism. This explains why this type of infection is often called the “hamburger disease”. Other reported vehicles of transmission are dairy products, contaminated water and more and more often vegetables. Produce can be contaminated in the pre-harvest phase by manure or water on the field or in the post-harvest phase during manipulation. Interhuman transmission has been often reported, but since humans do not present long-term carriage, the “sourcesink” model applies: humans represent a dead-end in the survival of the organism while cattle represent the source (26).


3

Acta Clinica Belgica, 2012; 67-? Sporadic and epidemic cases, worldwide

Verotoxins, Attachment-effacement phenotype

Aggregative adherence, heat-stable enterotoxin (EAST)

Invasion of colonic epithelial cells (similarly to Shigella spp.)

Diffuse adherence by fimbriae

VTEC (STEC) (EHEC)

EAEC

EIEC

DAEC

High

Moderate

Low

Low

Non human only

A

B

C

D

E

Not applicable

Rare

Rare

Uncommon

Common

Outbreaks

Poorly understood

Sporadic and epidemic cases in developing countries, rare in developed countries Watery diarrhoea

Not applicable

No

Yes

Yes

Yes

?

Human

Multiple

Multiple

O91:H21, O104:H4c, O104:H21, O113:H21, and others

O26:H11, O103:H2, O111:H-, O111:H8, O121:H19, O145:H-

O157:H7b, O157:H-

Serotypes

Watery, occasionally bloody diarrhoea

Human

Watery diarrhoea, often prolonged

Severe disease

Endemic and epidemic cases worldwide, traveler’s diarrhoea, persistent diarrhoea in HIV infection

Animal, mainly ruminants

Human (adhesins are species-specific)

Watery diarrhoea

Watery and bloody diarrhoea, haemorrhagic colitis, haemolytic uraemic syndrome

Human

Reservoir

Severe, prolonged non-bloody diarrhoea in infants

Symptoms

Adapted with permission from Karmali et al. (9). b The serotype of E. coli consists of a O-serogroup (lipopolysaccharide antigen) and a H-serogroup (flagellar antigen, absent in non-motile strains, designated as H-; each serotype presents non-motile variants, only the most frequent are presented here). c Not included in reference, added in this paper.

a

Relative incidence

Seropathotype

Table 2: Seropathotype classification of VTECa

Diarrhoea in young children in developing countries, traveller’s diarrhoea, diarrhoea in young piglets

Heat-labile (LT) and heat-stable (ST) enterotoxins, adhesins

ETEC

Outbreaks and sporadic cases in developing countries

EPEC adherence factor plasmid, Attachment-effacement phenotype

EPEC

Epidemiology

Main virulence factors

Acronym

Table 1: Main characteristics of diarrhoeagenic Escherichia coli

4 Infections with VTEC O157:H7, O104:H4 and others


Laboratory diagnosis

VTEC of serotype O157:H7 and its non-motile variant O157:H- possess a very particular phenotype, of which the most striking features are the absence of fermentation of sorbitol after 24 hours incubation, a negative b-glucuronidase reaction and resistance to tellurite. On basis of these properties, it can be easily detected by standard bacteriological techniques, for instance by culture on sorbitol-MacConkey agar supplemented with cefixime and tellurite (CT-SMAC) followed by agglutination for O157 antigen and biochemical identification (1). The reimbursement of this culture in cases of bloody diarrhoea or HUS is currently studied by the Health Insurance, RIZIV-INAMI. All other serotypes of VTEC, including sorbitolfermenting VTEC O157:H- that are rare outside Germany, are difficult to detect because they present the standard phenotype of commensal E. coli strains. Immunoassays for the detection of VT in enrichment broths can be used to detect these micro-organisms but the sensitivity is rather low and molecular techniques are preferred (1). In Belgium, the reference laboratory uses PCR on cultured colonies. Referral of stool samples of all HUS cases is important for the surveillance of these infections, as these cases can be considered as the “top of the iceberg”. VTEC concentrations in stools are high short after onset of diarrhoea but most patients will rapidly clear the organism. Some children can excrete it for weeks (27). Since onset of HUS occurs about one week after onset of diarrhoea, it is not always possible to isolate the agent. Some tests were developed to measure the antibodies response to different antigens, as for instance the lipopolysaccharide antigens determining the O serogroup, but this test is not fully evaluated and is reserved for research and epidemiological surveillance.

THE EPIDEMIC E. COLI STRAIN O104:H4, AN HYBRID OF VTEC AND EAEC PATHOTYPES Outbreak and epidemiological investigation

In May 2011, Germany reported an increased incidence of cases of bloody diarrhoea and HUS, associated with the isolation of VTEC of serotype O104:H4. Subsequently, this outbreak spread to other European countries and a few cases were imported in North America. On basis of the EU case definition and after exclusion of duplicates, 3910 probable and confirmed cases, including 782 HUS and 3128 non-HUS cases, were reported by 13 EU/EEA countries, in addition to 12 cases in Switzerland, USA and Canada (28, 29). The last onset date was 4 July. Most cases were seen in northern Germany. Thirty (30) HUS patients and 17 non-HUS patients died of which one patient in Sweden and one in the USA, all other deceases being observed in Germany, representing a mortality of 6.1% and 0.54%, respectively. All cases reported outside Germany reported a recent travel to this country or were infected through secondary spread from these cases, with the notable exception of 15 cases related to an event in Bègles, near Bordeaux in France (30). In comparison with other VTEC outbreaks, this epidemic presents special properties: a particular age and gender distribution, affecting mainly adult women, a high percentage of HUS cases of 25%, unexpected in this age category, and its large scale, rarely reached by VTEC O157:H7 and never by non-O157 serogroups (31). This unusual age and gender distribution may be related to dietary preferences of this

population group, as already observed during a large scale outbreak of VTEC O157:H7 associated with contaminated spinach in the United States (32). The source of infection was difficult to identify. On basis of the first case-control study, the first communications pointed out to leafy salad, tomatoes and cucumbers. Later, German authorities restricted their suspicion only to the latter and even cited a Spanish producer of cucumbers, causing an economical crisis in this sector. Finally, after a new case-control study, germinated seeds were identified as most plausible source. Although it was not possible to cultivate the bug, the observation that sprouts made with the same lot of fenugreek seeds imported from Egypt were used in Germany and at the event in Bègles confirmed the origin of the contamination. Fresh produce is increasingly recognized as vehicle of foodborne pathogens and is often consumed as a mixed food product, making it difficult to identify it as contamination source (33), as was the case here. Sprouted seeds were involved in multiple Salmonella and E. coli O157 infections. In particular, the massive outbreak of E. coli O157:H7 infection in school children in Sakai City, Japan, in 1996 was clearly related to contaminated radish sprouts, although all bacteriological tests on food and seeds remained negative (34). In their review on fresh fruit and vegetables as vehicle for the transmission of human pathogens, Berger et al. (33) underlined that the risk can be amplified after contamination in the pre-harvest phase by proliferation of pathogens during processing and post-harvest handling procedures. Water contaminated by run-off from nearby animal pastures or irrigation from a contaminated source can be an important source both in the field and in the post-harvest processing.

VT/EAEC O104:H4 strain

Very early during the outbreak, a VTEC of an unusual serotype, O104:H4, was identified in a few cases of HUS. Shortly thereafter, it was shown that this strain did not present the usual attachment/effacement properties of the most pathogenic VTEC, but the pathogenic determinants of enteroaggregative E. coli (EAEC), making it a mixed verotoxin-producing and enteroaggregative E. coli (designated here as VT/EAEC). Other characteristics were the production of Verotoxin/Shiga toxin VT2a (EQA nomenclature 2011, WHO Centre E. coli SSI, Copenhagen), presence of the aggR gene typical of EAEC as well as aggA encoding AAF/I adhesin associated with a strong ability to form biofilms but also iutA encoding aerobactin receptor, often found in extraintestinal pathogenic E. coli, the ter cluster conferring tellurite resistance, and irp2 and fyuA that are components of the iron uptake system on HPI (35, 36). Finally, a last unusual characteristic of this strain is the presence of a CTX-M15 extended-spectrum β-lactamase and resistance to co-trimoxazole (36). Strains combining these virulence factors were only exceptionally described before: a small cluster of HUS cases associated with serotype O111:H2 in France (37), one isolated case with serotype O86:H- in Japan (38) and a few other strains of serotype O104:H4 (35). It should be stressed, however, that other strains of this serotype did not harbour this combination of virulence genes but were either VTEC or EAEC. Very quickly after the outbreak brook out, several research groups used high-throughput DNA-sequencing technologies to compare the whole genome of the outbreak strain in comparison with other E. coli isolates of serotype O104:H4


5

6


and other EAEC isolates (39, 40, 41). The analysis of these sequences revealed that the outbreak strain is most related to other O104:H4 isolates. It is an enteroaggregative E. coli that acquired a verotoxin-encoding phage and an ESBLencoding plasmid. In our opinion, VT/EAEC O104:H4, originally not mentioned in the seropathotype classification represented in table 2 should now be classified in group 3: although it caused very severe disease, until now only one outbreak was reported and its incidence remains low.

CONCLUDING REMARKS Many questions remain open after the occurrence of this outbreak. How can the high virulence of this strain be explained? Is the enteroaggregative adherence associated with a higher uptake of VT from the intestine? Did other factors play a role? In particular, did the antibiotic resistance of this strain aggravate the clinical evolution? This factor could hasten the occurrence of HUS in patients receiving antibiotics, by eliminating the competing commensal flora without affecting the bug. Could such an outbreak happen again? One can speculate that the sources of infection of VT/EAEC are similar to those of EAEC. Unfortunately, this latter did not receive enough attention and its epidemiology is largely unknown. It is believed that it is spread in both developed and developing countries. No animal reservoir has been identified, showing the capacity to persist in human populations. It is probable that strains of EAEC could again acquire virulence genes of VTEC and result in such a strain, but it cannot be predicted if it will be as virulent and if it will be spread as in this outbreak. Finally, this incident stresses the importance of HUS surveillance, including efforts to cultivate VTEC isolates for further characterization and studies.

References 1. Nataro JP, Bopp CA, Fields PI, et al. Escherichia, Shigella, and Salmonella. In: Versalovic J, Caroll KC, Funke G, et al., eds. Manual of Clinical Microbiology. Washington, DC: ASM Press 2011; 603-626. 2. Kaper JB, Nataro JP, Mobley HL. Pathogenic Escherichia coli. Nat Rev Microbiol 2004; 2: 123-140. 3. Nataro JP, Kaper JB. Diarrhoeagenic Escherichia coli. Clin Microbiol Rev 1998; 11: 142-201. 4. Konowalchuk J, Speirs JI, Stavric S. Vero response to a cytotoxin of Escherichia coli. Infect Immun 1977; 18: 775-779. 5. O’Brien AD, LaVeck GD, Thompson MR, et al. Production of Shigella dysenteriae type 1-like cytotoxin by Escherichia coli. J Infect Dis 1982; 146: 763-769. 6. Riley LW, Remis RS, Helgerson SD, et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med 1983; 308: 681-685. 7. Karmali MA, Steele BT, Petric M, et al. Sporadic cases of haemolytic-uraemic syndrome associated with faecal cytotoxin and cytotoxin-producing Escherichia coli in stools. Lancet 1983; 1: 619-620. 8. Karmali MA, Gannon V, Sargeant JM. Verocytotoxin-producing Escherichia coli (VTEC). Vet Microbiol 2010; 140: 360-370. 9. Karmali MA, Mascarenhas M, Shen S, et al. Association of genomic O island 122 of Escherichia coli EDL 933 with verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease. J Clin Microbiol 2003; 41: 4930-4940. 10. Konczy P, Ziebell K, Mascarenhas M, et al. Genomic O island 122, locus for enterocyte effacement, and the evolution of virulent verocytotoxin-producing Escherichia coli. J Bacteriol 2008; 190: 5832-5840. 11. Tarr PI, Gordon CA, Chandler WL. Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet 2005; 365: 1073-1086. 12. Proesmans W. Typical and atypical hemolytic uremic syndrome. Kidney Blood Press Res 1996; 19: 205-208. 13. Piérard DCG, Proesmans W, Dediste A, et al. Hemolytic uremic syndrome in Belgium: incidence and association with verocytotoxin-producing Escherichia coli infection. Clin Microbiol Infect 1999; 5: 16-22.


14. Safdar N, Said A, Gangnon RE, et al. Risk of hemolytic uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 enteritis: a meta-analysis. JAMA 2002; 288: 996-1001. 15. Melton-Celsa AR, O’Brien AD. Structure, biology, and relative toxicity of Shiga toxin family members for cells and animals, p. 121-128. In J. B. Kaper and A. D. O’Brien (eds.), Escherichia coli O157:H7 and Other Shiga Toxin-Producing E. coli Strains. Washington D.C. ASM Press 1998; 121-139. 16. Bielaszewska M, Friedrich AW, Aldick T, et al. Shiga toxin activatable by intestinal mucus in Escherichia coli isolated from humans: predictor for a severe clinical outcome. Clin Infect Dis 2006; 43: 1160-1167. 17. Friedrich AW, Bielaszewska M, Zhang WL, et al. Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J Infect Dis 2002; 185: 74-84. 18. Orth D, Grif K, Khan AB, et al. The Shiga toxin genotype rather than the amount of Shiga toxin or the cytotoxicity of Shiga toxin in vitro correlates with the appearance of the hemolytic uremic syndrome. Diagn Microbiol Infect Dis 2007; 59: 235-242. 19. Frankel G, Phillips AD, Rosenshine I, et al. Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements. Mol Microbiol 1998; 30: 911-921. 20. Garmendia J, Frankel G, Crepin VF. Enteropathogenic and enterohemorrhagic Escherichia coli infections: translocation, translocation, translocation. Infect Immun 2005; 73: 2573-2585. 21. Aldick T, Bielaszewska M, Zhang W, et al. Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium. Microbes Infect 2007; 9: 282-290. 22. Griffin PM, Tauxe RV. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol Rev 1991; 13: 60-98. 23. Pierard D, Stevens D, Moriau L, et al. Isolation and virulence factors of verocytotoxin-producing Escherichia coli in human stool samples. Clin Microbiol Infect 1997; 3: 531-540. 24. De Schrijver K, Buvens G, Posse B, et al. Outbreak of verocytotoxin-producing E. coli O145 and O26 infections associated with the consumption of ice cream produced at a farm, Belgium, 2007. Euro Surveill 2008; 13. 25. Cobbaut K, Berkvens D, Houf K, et al. Escherichia coli O157 prevalence in different cattle farm types and identification of potential risk factors. J Food Prot 2009; 72: 1848-1853. 26. Pennington H. Escherichia coli O157. Lancet 2010; 376: 1428-1435. 27. Karch H, Russmann H, Schmidt H, et al. Long-term shedding and clonal turnover of enterohemorrhagic Escherichia coli O157 in diarrheal diseases. J Clin Microbiol 1995; 33: 1602-1605. 28. ECDC. Shiga toxin-producing E. coli (STEC): Update on outbreak in the EU (27 July 2011, 11: 00). In: http://ecdc.europa.eu/en/activities/sciadvice/Lists/ ECDC%20Reviews/ECDC_DispForm.aspx?List = 512ff74f%2D77d4%2D4ad8%2D b6d6%2Dbf0f23083f30&ID = 1166&RootFolder=%2Fen%2Factivities%2Fsciadvi ce%2FLists%2FECDC%20Reviews 2011. 29. WHO. Outbreaks of E. coli O10:H4 infection: update 30. In: http://www.euro.who. int/en/what-we-do/health-topics/emergencies/international-health-regulations/ news/news/2011/07/outbreaks-of-e.-coli-o104h4-infection-update-30 2011. 30. Gault G, Weill FX, Mariani-Kurkdjian P, et al. Outbreak of haemolytic uraemic syndrome and bloody diarrhoea due to Escherichia coli O104:H4, south-west France, June 2011. Euro Surveill 2011; 16. 31. Frank C, Werber D, Cramer JP, et al. Epidemic Profile of Shiga-Toxin-Producing Escherichia coli O104:H4 Outbreak in Germany - Preliminary Report. N Engl J Med 2011. 32. Wendel AM, Johnson DH, Sharapov U, et al. Multistate outbreak of Escherichia coli O157:H7 infection associated with consumption of packaged spinach, August-September 2006: the Wisconsin investigation. Clin Infect Dis 2009; 48: 1079-1086. 33. Berger CN, Sodha SV, Shaw RK, et al. Fresh fruit and vegetables as vehicles for the transmission of human pathogens. Environ Microbiol 2010; 12: 2385-2397. 34. Michino H, Araki K, Minami S, et al. Massive outbreak of Escherichia coli O157:H7 infection in schoolchildren in Sakai City, Japan, associated with consumption of white radish sprouts. Am J Epidemiol 1999; 150: 787-796. 35. Scheutz F, Moller NE, Frimodt-Moller J, et al. Characteristics of the enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli O104:H4 strain causing the outbreak of haemolytic uraemic syndrome in Germany, May to June 2011. Euro Surveill 2011; 16. 36. Bielaszewska M, Mellmann A, Zhang W, et al. Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study. Lancet Infect Dis 2011. 37. Morabito S, Karch H, Mariani-Kurkdjian P, et al. Enteroaggregative, Shiga toxinproducing Escherichia coli O111:H2 associated with an outbreak of hemolyticuremic syndrome. J Clin Microbiol 1998; 36: 840-842. 38. Iyoda S, Tamura K, Itoh K, et al. Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia coli O86:HNM isolated from patients. FEMS Microbiol Lett 2000; 191: 7-10. 39. Mellmann A, Harmsen D, Cummings CA, et al. Prospective Genomic Characterization of the German Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing Technology. PLoS One 2011; 6: e22751. 40. Rasko DA, Webster DR, Sahl JW, et al. Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany. N Engl J Med 2011; 365: 709-717. 41. Rohde H, Qin J, Cui Y, et al. Open-source genomic analysis of Shiga-toxin- producing E. coli O104:H4. N Engl J Med 2011; 365: 718-724.