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Enterohaemorrhagic E. coli (EHEC) is a bacterium that can cause severe foodborne disease
Enterohaemorrhagic E. coli (EHEC) is a bacterium that can cause severe foodborne disease

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J. Vet. Med.

B 53, 299–305 (2006) Ó 2006 The Author Journal compilation Ó 2006 Blackwell Verlag, Berlin ISSN 0931–1793

National Reference Laboratory for Escherichia coli, Federal Institute for Risk Assessment, Berlin, Germany

Emerging Enterohaemorrhagic Escherichia coli, Causes and Effects of the Rise of a Human Pathogen
L. Beutin
Address of author: National Reference Laboratory for Escherichia coli (NRL-E.coli), Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, D-12277 Berlin, Germany; Tel.: +49 30 8412 2259; fax: +49 30 8412 2983; E-mail: l.beutin@bfr.bund.de Received for publication November 10, 2004

Shiga toxin (Stx) [Verotoxin (VT)]-producing Escherichia coli (STEC), also called enterohaemorrhagic E. coli or VTEC are emerging zoonotic agents and became most important as human pathogens, particularly in the industrialized countries. Production of cytotoxins, also called Stx or VT, is the major pathogenicity determinant of STEC, which can cause life-threatening haemorrhagic diseases in humans. The spectrum of STEC phenotypes is diverse and domestic and wildlife animals constitute important reservoirs for these bacteria. STEC are spread from animal faeces to the environment, water and food. Ingestion of contaminated foodstuff and water, as well as contact with the environment, STEC-excreting animals or humans are the major sources of human infection. Economical changes in animal and food production, alteration of consumer habits and lack of specific immune response, particularly in urbanized populations, have contributed to the recent spread of STEC as a zoonotic agent. Supranational surveillance networks as well as national reference laboratories as sentinels play an important role in the prevention and control of STEC infections in humans. Development of new vaccines and probiotics may serve as future tools to control the spread of STEC in animals and humans.

Among the pathogenic Escherichia coli types, enterohaemorrhagic E. coli (EHEC) became most important as human pathogens, particularly in the industrialized countries with moderate climate. The rapid increase of human EHEC infections reported in the last decades has alarmed public health authorities and has raised a debate on the microbiological safety of food of animal origin, which is often incriminated as source of EHEC transmission to humans. The reasons for the recent emergence and rapid spread of EHEC infections in different parts of the world were investigated for different aspects and are far from being fully recognized. Current data indicate that manifold factors contribute to the change in emergence of these bacterial pathogens and the aim of this review is to present some of the most important criteria which could have contributed to the spread of these pathogens.

Enterohaemorrhagic E. coli were defined as a subgroup of Shiga toxin (Stx)-producing E. coli (STEC) which were
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known to cause haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) in humans (Levine, 1987). The EHEC prototype strain, E. coli O157:H7, became first known as a human pathogen in 1982 as causative agent in foodborne-outbreaks associated with consumption of undercooked beef (Riley et al., 1983). EHEC were defined as STEC showing similar clinical, epidemiological and pathogenic features as the EHEC prototype strain, E. coli O157:H7 (Levine, 1987). Later, the term EHEC was used for those STEC which have been demonstrated to cause diarrhoea in humans (Anonymous, 1997) and it was proposed to divide EHEC into ÔtypicalÕ and ÔatypicalÕ according to the presence of other virulence attributes such as the eae gene for bacterial intimate adherence and the plasmid located E-hlyA gene encoding production of enterohaemolysin (Nataro and Kaper, 1998). The division of Stx-producing strains into STEC and EHEC is not very clear-cut, because it is not proved if any of the naturally occurring STEC strains are apathogenic for humans. To avoid confusion in nomenclature, the term STEC will be used for all types of Stx-producing E. coli in this review. On the other hand, it is certain that STEC strains show considerable differences regarding their virulence and pathogenicity. The absence of virulence markers such as the eaegene does not necessarily indicate that these strains possess a lower virulence for humans (Paton et al., 1999). Apart from STEC O157:H7, certain other STEC types which represent clonal groups, such as motile and non-motile strains of O26:[H11], O103:H2, O111:[H8] and O145:[H28] were associated with increased virulence for humans and were more frequently associated with HC and HUS than other STEC types. Besides this well known Ôgang of fiveÕ, other emerging highly virulent STEC types such as O118:[H16] and O121:[H19] were identified more recently (Maidhof et al., 2002; Tarr et al., 2002). The continuous evolution of these pathogens is triggered by selective pressure from the host immune system and from the environment. It was demonstrated that STEC undergo frequent genetic rearrangements in their chromosome, their virulence plasmids, stx-phages and in the LEE pathogenicity islands coding for the attaching and effacing (A/E) phenotype (Brunder et al., 1999; Beutin et al., 2005; Garrido et al., 2006). The future emergence of new genetic variants of highly virulent STEC clones is therefore very likely. www.blackwell-synergy.com



L. Beutin the only way to identify all types of STEC in any kind of sample (Bettelheim and Beutin, 2003). However, not all of surveillance systems established worldwide are based on detection of Stx or the underlying genes and some national surveillance programmes are more restricted only to identification of STEC O157, which is carried out by screening for phenotypical traits such as serotype and absence of sorbitol fermentation.

Virulence Factors
Shigatoxin-producing E. coli and EHEC are characterized by production of Stx also called Verotoxins (VT). STEC (VTproducing E. coli) strains can produce one or more different types of Stx. Stx are N-glycosidases, enzymes which inhibit protein synthesis in eucaryotic cells leading to cell death, tissue damage and organ failure (Paton and Paton, 1998). The genes for production of major Stx-types, called Stx1 (VT1) and Stx2 (VT2), are located on the genome of temperate lambdoid bacteriophages, which are integrated in the chromosome of the bacteria. Stx phages can be induced to the lytic cycle and free phages can infect other bacteria resulting in horizontal gene transfer of stx genes to E. coli and other Enterobacteriaceae as shown by in vivo and in vitro experiments (Herold et al., 2004). As a consequence, the diversity of E. coli strains which are associated with production of Stx is high and new STEC types are still detected. Today, more than 200 E. coli serotypes representing numerous STEC-clones are described (http:// www.microbionet.com.au/vtectable.htm). Since the first description of Stx, a growing number of genetic variants of the prototype toxins Stx1 and Stx2 have been described (Scheutz et al., 2001). Human diseases caused by STEC show a broad spectrum, ranging from mild diarrhoea to HC and HUS. Primarily the production of Stx2 as well as the presence of the eae gene encoding intimate adherence of the bacteria to the intestinal mucosa were associated with increased virulence of STEC producing strains and with severe clinical manifestation (Boerlin et al., 1999; Friedrich et al., 2002, 2003; Beutin et al., 2004; Ethelberg et al., 2004). Other toxin variants, such as Stx1c, Stx2-O118 and Stx2e were associated with more uncomplicated cases of diarrhoea (Friedrich et al., 2002, 2003) and newly described toxin types such as Stx1d, Stx2g and Stx2-NV206 have not yet been explored for their role in human pathogenicity (Bertin et al., 2001; Burk et al., 2003; Leung et al., 2003).

Type Spectrum
Remarkable differences were observed in the type spectrum of STEC isolated from humans in different countries. One explanation could be that the identification and isolation methods used in diagnostic laboratories differ largely for sensitivity and specificity. Sporadic infections with non-O157 STEC types are frequent in many countries but are often not detected due to inadequate testing methods. As a consequence surveillance of these pathogens is less developed than for STEC O157 (Mead et al., 1999; Anonymous, 2006). Only a few reference laboratories worldwide have established the methodologies for complete serotyping of O and H antigens of E. coli and it can be assumed that only a minor fraction of STEC isolates from routine diagnostic laboratories are investigated for their serotypes and their virulence attributes. This practice may result in delayed or in lack of detection of new emerging human pathogenic STEC types with the consequence that adequate prevention measures are not taken in a timely fashion. The role of E. coli reference laboratories has proved to be crucial in detection of human infections with sorbitol-nonfermenting STEC O157:H7 in the USA in 1982 and 10 years later in detection of sorbitol-fermenting STEC O157 in Germany (Riley et al., 1983; Gunzer et al., 1992). Despite the bias resulting from the use of STEC detection methods, which are not well comparable for their specificity and sensitivity, there is a considerable difference between countries for the frequency of human cases infected with these agents and for the predominant types of STEC in different geographical regions. The investigation of the animal reservoir for the presence and type spectrum of STEC is important to monitor trends of emergence of known and new types of STEC, which can cause infections in humans. As an example, STEC O118:[H16] became first prevalent in healthy and diarrhoeic cattle in Germany in 1989 and have emerged as human pathogens since 1996. Most of the human infections were associated with a rural environment and direct transmission from animals to humans was demonstrated (Wieler et al., 1996; Beutin et al., 2000). STEC isolates from cattle and humans were found to be genetically closely related belonging to a clonal type which is widespread in Europe and South America (Maidhof et al., 2002; de Castro et al., 2003).

Disease Spectrum
The magnitude and the severity of STEC infections in humans and the impact of these pathogens on public health was the subject of four international WHO consultations on foodborne and zoonotic aspects and on prevention and control between 1994 and 1998 (Anonymous, 1995, 1996, 1997, 1998). The spectrum of disease caused by different STEC is varying and human asymptomatic carriers as well as long-term excretors are not infrequent in adults who were in contact with STECinfected patients. Children and the elderly were identified as major risk groups for developing severe disease such as HUS and thrombotic thrombocytopenic purpura as sequelae from STEC infections (Taylor and Monnens, 1998). In consequence, surveillance of enteric HUS was established in many countries since it was shown to be a reliable indicator for STEC infections in the human population. HUS is the major cause of acute renal failure in children and >90% of HUS cases are caused by infections with STEC (Mahon et al., 1997). Although enteric infections with STEC are today notifiable in many countries their surveillance is still limited due to suboptimal detection systems and in consequence to underreporting. Epidemiological studies from different countries have shown that diverse types of STEC may infect and cause illness in humans and detection of stx genes or production of Stx is

Animal and Environmental Reservoir
Epidemiological analysis of outbreaks with EHEC O157 revealed cattle as a major animal reservoir for these pathogens (Karmali, 1989). Until today, an increasing number of domestic and wildlife animal species, in particular ruminants, have been identified as natural reservoirs and sources of STEC infections (Caprioli et al., 2005). Over the last two decades, STEC were identified in almost all geographical regions of the

Emerging and Rapid Spread of EHEC Infections world. Direct and indirect animal contact was identified as important transmission route for STEC in the environment followed by person to person spread of STEC as second most important way of transmission (Anonymous, 1997; Mead et al., 1999). As a consequence of faecal shedding of STEC by animals, the degree of environmental contamination with these bacteria is important. Studies have shown that EHEC O157 may persist as viable bacteria in animal faeces over more than 20 months and contamination of soil with effluents from agriculture, abattoirs and sewage water is important particularly in areas with high density of cattle and other domestic animals (Maule, 2000). Contamination of farmland soil associated with contamination of surface and drinking water resulted in human infections from environmental sources as demonstrated in numerous cases (Williams et al., 2005). Water-borne outbreaks have gained importance and count for 9% of 350 outbreaks in the USA, which were notified within a 20-years time period (Rangel et al., 2005). Extreme weather conditions such as heavy rainfall and elevated air temperatures were related to increased water-borne outbreaks with STEC O157 and other bacteria (Thomas et al., 2006) and STEC infections were generally found to increase in the warm season (Barkocy-Gallagher et al., 2003; Anonymous, 2004). Environmental contamination of farmland and irrigation water with STEC explains their finding in vegetables, and consumption of STEC O157 contaminated radish sprouts accounted for the world’s largest outbreak in Sakai City, Japan in 1996, with more than 6000 cases of illness (Michino et al., 1999). The dimension of environmental contamination with STEC becomes apparent by the presence of these agents in shellfish collected in coastal areas polluted with effluents from farmland (Gourmelon et al., 2006). Studies on dissemination of STEC on farms demonstrated the presence of the agent at multiple sites in the farm environment (Schouten et al., 2005). Farm and wildlife animals such as insects, rodents and wild birds may serve as possible vectors of transmission (Ejidokun et al., 2006).

301 certainly be very useful for prevention of STEC entry in the food chain. Shedding of STEC from animals was found to be influenced by many factors such as animal age, diet, stress, housing conditions and season. Longitudinal studies on STEC excretion by animals have shown that resident types of STEC strains such as O116:H21 in cattle can be excreted over months by the same animal and are widespread within animal herds probably due to transmission from animal to animal (Beutin et al., 1997). Typical resident STEC strains from cattle or sheep appear to be quite specific for their animal host and are rarely isolated from other animals or human patients, in contrast to human pathogenic STEC types which show a broad host range and are therefore found in different animal species including man (Trevena et al., 1996; Leomil et al., 2005). Typical human pathogenic STEC do not normally represent the major STEC flora of animals but can exceptionally become the prominent type in herds and individual animals as Ôsuper-sheddersÕ. The reasons for these findings are not well known, but future strategies should investigate the possibility of using animal host-specific STEC strains as probiotics in animals for possible prevention of colonization with human virulent STEC types such as O157:H7.

Prevalence in Humans
Prevalence rates and types of STEC found in human patients vary also between different regions in the same country, as reported from the UK (Anonymous, 2004) Canada and the USA. In the USA, incidence rates for O157 are higher in the northern than in the southern states which could be explained with large rural populations in the northern States and increased contact to farm animals (Fey et al., 2000). High cattle density and application of manure to the surface of farmland was related to high incidence of STEC infections in humans (Valcour et al., 2002). On the other hand, the elevated exposure of the rural population to STEC compared with urban residents has resulted in an elevated seroresponse to Stx1 and Stx2 in the rural population and the presence of anti-shigatoxin antibodies was suggested to play a role in protective immunity (Karmali et al., 2003). Acquired immunity might therefore play an important role for prevention of STEC infections in early childhood. As a result from a Canadian study, many dairy farm residents experience subclinical immunizing STEC infections at a young age, which frequently involve non-O157 STEC found in cattle (Wilson et al., 1996). Besides Stx, intimin (eae) plays a major role in the virulence of human pathogenic STEC and also in Stx-negative enteropathogenic E. coli (EPEC), and eae-positive STEC are associated with high pathogenicity for humans (Boerlin et al., 1999). In patients, a strong relationship was found between infections with intimin-positive STEC, young age, and severe clinical picture. In contrast, adult patients were more frequently infected with eae-negative STEC with a milder clinical outcome (Beutin et al., 2004). Modified intimin was therefore proposed as a candidate for vaccination of humans against virulent STEC types as it worked in an experimental animal model (Agin et al., 2005). In regions where EPEC expressing intimin and LPS similar to those of STEC are endemic, antiintimin and anti-LPS antibodies are found in the human population indicating cross-reactive immunity to EPEC and STEC (Ghaem-Maghami et al., 2001; Palmeira et al., 2005).

Epidemiological studies have shown that STEC are present in numerous serotypes in cattle and other domestic animals worldwide, independent of geographical regions, animal race, husbandry and climate. The close association of cattle and other ruminants with STEC points to an ecological role of STEC in domestic and wildlife ruminants which may be explained more by symbiosis than by simple parasitism. Elucidation of the ecological role of STEC in ruminants would contribute to understand the nature of the animal reservoir and help to develop future prevention strategies. Differences between countries, regions, farms and animal herds were found in regard to prevalence and shedding of highly virulent STEC types such as O157:H7, O26:[H11] and others. For O157, so-called super-shedding animals were found in studies on cattle herds in Scotland and it was calculated that 80% of transmission arises from 20% of most infective individual animals (Matthews et al., 2006a). Only 2% of Scottish farms showed high incidence of O157 shedding animals compared to 78% of Scottish cattle farms that were negative for O157 (Matthews et al., 2006b). Early detection of super-shedding animals and a better understanding of the mechanisms which convert individual animals to this state will

302 These findings might explain the differences in susceptibility to STEC as found in human populations from different geographical regions. The increase of STEC infections in humans in the industrialized countries of the northern hemisphere was concomitant with the decrease of infections with classical EPEC at the same time period and could be explained by loss of cross-reacting protective immunity in the human population. Cross-protective immunity was found in humans from countries where EPEC incidence rates are still high such as Brazil (Palmeira et al., 2005). The low incidence of STEC in human infections in Brazil and other developing countries is in contrast to the high incidence of STEC found in domestic animals from the same region which is comparable with that of the industrialized countries (Kaddu-Mulindwa et al., 2001; Irino et al., 2005). These findings underline that animals constitute a major reservoir for STEC worldwide and the different frequencies of human infections with STEC point to the immune status of the human host as a major factor for susceptibility.

L. Beutin

Causes of STEC Emergence
In the first years of STEC surveillance, it was reported that the frequency of STEC infections in humans is continuously increasing. To some extent, this finding is due to the establishment of more sophisticated STEC detection and isolation methods and to increased surveillance of these pathogens resulting in more identified clinical cases of STEC infections. Although there is no doubt that STEC have played a role in animals and humans since a long time (Beutin and Muller, 1998; Bettelheim and Beutin, 2003), it is obvious that these pathogens became more prevalent in humans in recent years. Different factors may be taken into account for this phenomenon: in parts, the industrialization of agriculture has resulted in mass production of animals and has led to an increase of domestic animal densities in larger geographical regions. As a consequence of that, the rate of STEC infections in humans which correlates positively with cattle density is elevated (Michel et al., 1999; Valcour et al., 2002; Kistemann et al., 2004; Haus-Cheymol et al., 2005). A second point is the consumption of meat and milk products, that has steadily increased in the industrialized countries in which STEC infections have gained in importance. Meat and milk account most frequently among foodstuff which were incriminated as sources in foodborne outbreaks with STEC in different countries (Anonymous, 1997; Rangel et al., 2005). Moreover, consumer habits in the industrialized countries have changed towards increased consumption of raw vegetables as well as to consumption of undercooked meat, conditions which may favour foodborne infections. A third factor which might have an influence to the spread of STEC in nature is the increased application of antibiotics in agriculture for nutrition and therapy in animal mass production (Kohler et al., 2000). A number of antimicrobial agents were not only shown to enhance Stx production in the bacteria but also to induce stxphages to the lytic cycle resulting in the release of phages into the environment. Free bacteriophages were found to be more stable against environmental stress than their bacterial host (Moce-Llivina et al., 2003) and present an important reservoir for generation of STEC strains by phage transduction which can occur in the environment as well as in the mammalian host (Kohler et al., 2000; Zhang et al., 2000; Toth et al., 2003). Moreover, a large reservoir of so-called atypical EPEC strains is present in animals and humans. Some of these strains are genetically highly similar to STEC showing the same serotypes and virulence markers but lack stx genes which can be acquired by horizontal gene transfer (Leomil et al., 2005). Taken together, these findings underline the dynamics of STEC evolution in nature and discourage from strategies which point to eradication of STEC in their natural reservoirs.

Laboratory methods used for detection of STEC can differ widely even within the same country leading to a considerable bias in detection rates particularly with non-O157 STEC strains (Combs et al., 2005). However, global scientific effort aims to standardize detection and surveillance of all STEC independent of their serotype, as traditionally performed in Germany and other European countries, and is now adopted in countries which traditionally have focused only on STEC O157:H7 (Brooks et al., 2005). The Foodborne Diseases Active Surveillance Network (FoodNet) of the CDC has started collecting data on non-O157 STEC infections since 2000 as well as comprehensive information on HUS (Anonymous, 2006). International networks such as Enter-Net and Med-Vet-Net have shown to play an important role in harmonization of detection and reporting protocols and for launching concerted action programmes for research and surveillance of STEC in many European countries. Data from industrialized countries with well established nationwide reporting systems for STEC O157 indicate that the increase of STEC infections in humans has slowed down. Recent data from the UK indicate that the number of reported cases of STEC O157 has stabilized to approximately 1000 per year since 1995 (Anonymous, 2004). Numbers of O157 outbreaks per year have stabilized in the USA since 1998 and the median size of these outbreaks was significantly reduced since 1991 (Rangel et al., 2005). These findings could indicate that the concept of hazard analysis and critical control points (HACCP) in food production and control measures for prevention and containment of outbreaks such as PulseNet have already taken action (Gerner-Smidt et al., 2005). Surveillance programmes such as PulseNet have been established in the USA and within the Med-Vet-Net in Europe and will be effective for control of international dissemination of STEC and other microbial pathogens. It has to be shown in future, if trends observed for STEC O157 are also applicable to non-O157 STEC infections which are less well monitored in many regions.

Both, economical changes in animal and food production including international trade, mass catering and changing consumer habits as well as environmental factors are shown to be closely associated with the emergence of STEC as human pathogens. Strategies for prevention of human infections with these bacteria should not only based on technical solutions such as HACCP concepts to minimize STEC contamination in all stages of the food chain but should also consider the ecological role of these bacteria in animals.

Emerging and Rapid Spread of EHEC Infections Development of vaccines directed against STEC colonization factors such as intimin as well as adoption of probiotics, which inhibit the colonization of animals with human pathogenic STEC types could serve as new tools in the struggle against these pathogens. The international harmonization of STEC detection systems, surveillance and reporting as well as the establishment of specific reference laboratories in all countries will contribute to prevention and control of STEC infections in humans worldwide. Last not least, the immune status of the human host plays a major role in susceptibility to infections with STEC and other pathogens and nutritional factors which could have an influence on the natural resistance of humans and animals should be part of future investigations.

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