Escherichia coli O157:H7: animal reservoir and sources of human infection

This review surveys the literature on carriage and transmission of enterohemorrhagic Escherichia coli (EHEC) O157:H7 in the context of virulence factors and sampling/culture technique. EHEC of the O157:H7 serotype are worldwide zoonotic pathogens responsible for the majority of severe cases of human EHEC disease. EHEC O157:H7 strains are carried primarily by healthy cattle and other ruminants, but most of the bovine strains are not transmitted to people, and do not exhibit virulence factors associated with human disease. Prevalence of EHEC O157:H7 is probably underestimated. Carriage of EHEC O157:H7 by individual animals is typically short-lived, but pen and farm prevalence of specific isolates may extend for months or years and some carriers, designated as supershedders, may harbor high intestinal numbers of the pathogen for extended periods. The prevalence of EHEC O157:H7 in cattle peaks in the summer and is higher in postweaned calves and heifers than in younger and older animals. Virulent strains of EHEC O157:H7 are rarely harbored by pigs or chickens, but are found in turkeys. The bacteria rarely occur in wildlife with the exception of deer and are only sporadically carried by domestic animals and synanthropic rodents and birds. EHEC O157:H7 occur in amphibian, fish, and invertebrate carriers, and can colonize plant surfaces and tissues via attachment mechanisms different from those mediating intestinal attachment. Strains of EHEC O157:H7 exhibit high genetic variability but typically a small number of genetic types predominate in groups of cattle and a farm environment. Transmission to people occurs primarily via ingestion of inadequately processed contaminated food or water and less frequently through contact with manure, animals, or infected people.     

Human infections with non-O157 Shiga toxin-producing Escherichia coli, Switzerland, 2000-2009

We characterized 97 non-O157 Shiga toxin (stx)-producing Escherichia coli strains isolated from human patients during 2000-2009 from the national reference laboratory in Switzerland. These strains belonged to 40 O:H serotypes; 4 serotypes (O26:H11/H-, O103:H2, O121:H19, and O145:H28/H-) accounted for 46.4% of the strains. Nonbloody diarrhea was reported by 23.2% of the patients, bloody diarrhea by 56.8%. Hemolytic uremic syndrome developed in 40.0% of patients; serotype O26:H11/H- was most often associated with this syndrome. Forty-five (46.4%) strains carried stx2 genes only, 36 strains (37.1%) carried stx1, and 16 (16.5%) strains carried stx1 and stx2. Genes encoding enterohemolysin and intimin were detected in 75.3% and 70.1% of the strains, respectively. Resistance to ≥1 antimicrobial agent was present in 25 isolates. High genetic diversity within strains indicates that non-O157 stx-producing E. coli infections in Switzerland most often occurred as single cases.     

Investigation of human infections with verocytotoxin-producing strains of Escherichia coli (VTEC) belonging to serogroup O118 with evidence for zoonotic transmission

Twenty verocytotoxigenic Escherichia coli (VTEC) O118 strains isolated between 1996 and 1998 from human patients in Germany were analysed for their serotypes, their virulence markers and their epidemiological relatedness. Three strains were typed as O118:H12, these carried only the VT2d-Ount variant gene and were not associated with diarrhoea or haemolytic uraemic syndrome (HUS). Seventeen strains were serotyped as O118:H16 or O118:non-motile (NM). These carried all the genes for VTI, eae and EHEC-haemolysin. The O118:H16/NM strains were from diarrhoea (13 cases) and HUS (2 cases). Sixteen of the patients were young infants and most infections were associated with a rural environment. Evidence for zoonotic transmission from cattle to humans was found in two cases. The epidemiological relationship between the human and bovine O118:H16/NM isolates was indicated by homogeneous plasmid patterns and by very similar XbaI restriction patterns obtained by pulsed-field gel electrophoresis. VTEC O118:H16/NM are emerging pathogens in Germany and should be classified as new enterohaemorrhagic E. coli (EHEC) types.     

Escherichia coli O104 Associated with Human Diarrhea, South Africa, 2004-2011

To determine the origin of >4,000 suspected diarrheagenic Escherichia coli strains isolated during 2004-2011 in South Africa, we identified 7 isolates as serotype O104; 5 as enteroaggregative E. coli O104:H4, and 2 as enteropathogenic E. coli O104:non-H4. Pulsed-field gel electrophoresis showed that these isolates were unrelated to the 2011 E. coli O104:H4 outbreak strain from Germany.     

Infektionen des Menschen durch enterohämorrhagischeEscherichia coli (EHEC) in Deutschland, 1996

Zusammenfassung  1996 wurden von uns insgesamt 96 F?lle von h?molytischur?mischem Syndrom (HUS) durch EHEC Erregernachweis und/oder Nachweis spezifischer Antik?rper gesichert. Hinzu kommen 63 durch EHEC verursachte Enteritisf?lle und 40 Ausscheider. Neben den bereits in den Vorjahren geh?uft nachgewiesenen EHEC O157 (O157:H7, O157:H⁻), O26 (O26:H⁻, O26:H11) und O111 (O111:H⁻, O111:H2) ist 1996 erstmals EHEC O103:H2 als Erreger von HUS- und Enteritisf?llen aufgetreten und wurde zweith?ufigster Erreger von EHEC-Infektionen des Menschen. Insgesamt wurden bei 149 komplett charakterisierten Isolaten 26 verschiedene Serovare und zus?tzlich elf St?mme mit nichttypisierbarem O-Antigen nachgewiesen. Dies unterstreicht einerseits die Notwendigkeit der EHEC-Isolierung auf der Grundlage des Shiga Toxin-(Stx)-Nachweises bzw. der stx-Determinanten, anderersteis eine weitere Subtypisierung der Isolate (u. a. Serotypisierung). Ein interdisziplin?res Agieren auf gesundheitspolitischer, medizinischer, veterin?rmedizinischer und administrativer Ebene ist für Deutschland unbedingt zu fordern, um Gesundheits- und Verbraucherschutz zu optimieren.

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Human infections due to enterohaemorrhagic Escherichia coli (EHEC) in Germany, 1996

Summary  In 1996 we confirmed a total of 96 cases of hemolytic uremic syndrome (HUS) by identification of the enterohemorrhagic E. coli (EHEC) pathogen and/or detection of specific antibodies. In addition there were 63 cases of EHEC-induced enteritis and 40 asymptomatic carriers of the organism. Alongside EHEC O157 (O157:H7, O157:H⁻, O26 (O26:H⁻, O26:H11) and O111 (O111:H⁻, O111:H2), which have already been identified frequently in previous years, in 1996 EHEC O103:H2 appeared for the first time as a causative pathogen of HUS and cases of enteritis and was the second most common pathogen causing EHEC-infections in the human. Out of a total of 149 fully characterised isolated, 26 different serovars were identified along with eleven strains with nontypable O-antigens. These findings underline on the one side the importance of EHEC-isolation by identification of the Shiga toxin- (Stx) or stx determinants, and on the other the need for further subtyping of the isolates (e.g. serotyping). An inter-disciplinary effort involving governmental health authorities, medical and veterinary bodies and administration departments in Germany must be called for in order to optimise health protection and the safety of the consumer.

     

A long-term study on the prevalence of shiga toxin-producing Escherichia coli (STEC) on four German cattle farms

The occurrence of Shiga toxin-producing Escherichia coli (STEC) was studied on four cattle farms. STEC were detected in 29-82% of the cattle. STEC with additional EHEC markers were detected on all farms. The occurrence of the complete virulence marker pattern (stx1 and/or stx2, eae, EHEC(hlyA), katP, espP) was correlated with the presence of known STEC serotypes. STEC O26:H11 and O165:H25 with the complete pattern of virulence markers were the most prevalent. STEC O157 (H7/H-) STEC O103:H2 and STEC O145:H- were found sporadically. Five clonal subgroups of the STEC O26:H11 isolates were identified by pulsed-field gel electrophoresis. STEC O26:H11 were present in three groups of cattle. This serotype was detected in a single group over the entire fattening period. Most STEC O26:H11 with the complete pattern of potential virulence markers were found in clinically healthy cattle. These animals may represent a risk factor for farmers and consumers.     

Cross reactivity of enterohemorrhagic Escherichia coli O157:H7-specific sera with non-O157 serotypes

Escherichia coli O157:H7 is an important food- and water-borne pathogen of humans, causing Hemorrhagic Colitis and Haemolytic Uremic Syndrome. Colonization of both cattle and human hosts is mediated through the action of effector molecules secreted via a Type III secretion system, a mechanism shared by other enterohemorrhagic E. coli (EHEC). We recently reported that vaccination of cattle with Type III-secreted proteins (TTSPs) resulted in decreased shedding of the organism following both experimental infection as well as under conditions of natural exposure. In order to extend this to non-O157 EHEC serotypes, we examined the serological cross reactivity of TTSPs of serotypes O26:H11, O103:H2, O111:NM and O157:H7. Western blotting experiments with polyclonal antisera directed against serotype O157:H7 TTSPs suggested that there was significant cross reactivity, although there was limited cross reactivity when two Tir- and EspA-specific monoclonal antibodies were used. Groups of cattle were then vaccinated with TTSPs produced from each of the above serotypes and the magnitude and specificity of the responses were measured. All animals responded well with antibodies to TTSPs of the homologous serotype. However, limited cross reactivity was observed against the others. No cross reactivity was observed against Tir and EspA of serotype O157:H7. These results suggest that vaccination of cattle with TTSPs as a means of reducing the risk of EHEC transmission to humans will induce protection that is serotype specific.     

Prevalence and characteristics of Escherichia coli serotype O157:H7 and other verotoxin-producing E. coli in healthy cattle.

From February to July of 1994, 328 faecal samples from 32 herds were collected and verotoxin-producing Escherichia coli (VTEC) found on 84% of the farms. The proportion of animals infected varied from 0-63%. VTEC were recovered from 52 (20%) of 257 cows and from 16 (23%) of 71 calves. Although the VTEC belonged to 25 different serogroups, 7 (O8, O20, O22, O77, O113, O126 and O162) accounted for 46% of strains. Nearly 45% of the strains. Nearly 45% of the 83 bovine VTEC strains belonged to serogroups associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. However, only 2 (2%) of 83 VTEC strains isolated from cattle belonged to enterohaemorrhagic E. coli (EHEC) serotypes (O26:H11 and O157:H7), and only 8 (10%) were positive for the attaching and effacing E. coli (eae) gene sequence. Polymerase chain reaction (PCR) showed that 17 (20%) of VTEC strains carried VT1 genes, 43 (52%) possessed VT2 genes, and 23 (28%) carried both VT1 and VT2 genes. Characterization of VTEC isolates revelated a heterogeneous population in terms of serogroup and toxin type in the positive herds. This study confirms that healthy cattle are a reservoir of VTEC, but, the absence of eae genes in most bovine VTEC strains suggests that they may be less virulent for humans than eae-positive EHEC.     

Infektionen des Menschen durch enterohämorrhagische Escherichia coli (EHEC) in der Bundesrepublik Deutschland von 1998 bis 2001 Prävalenz und Typenspektrum

Intestinal infections in Germany due to enterohemorrhagic E. coli bacteria (EHEC) between 1998 and 2001 reveal a large scale of biological diversity of their pathogens. However, no dramatic increase of their clinical importance and public health implications has been observed. As strains of serovar O157:H7 have continuously declined as causative agents, other serovars such as O26:H11 and O103:H2 have replaced them. The great diversity of the EHEC pathogens might point to a great number of various infection routes and sources. Since recently new pathogenic factors of EHEC bacteria have been detected (especially by the sequencing of the genome of EHEC), it is currently not possible to define a clear-cut difference between human pathogens and nonhuman pathogens. The enhanced surveillance of EHEC pathogens with respect to their biological diversity and dynamics, their epidemic spread, and their infection routes and sources remain an essential task of the public health authorities.     

Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx2c, either alone (16%) or in combination with stx1 (74%) or stx2 (3%). PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.     

肠道致病菌暴发事件中分子分型技术的应用

目的探讨建立快速敏感的肠道病原菌的诊断分型方法。方法2002年7月用脉冲场电泳胶（PFGE）与噬菌体分型实验方法对发生在日本静冈县的4起暴发事件中29株宋内志贺菌（Shigella.sonnei）分离株与1999年口本静冈县某区现场分离的12株肠出血性大肠埃希菌（enterohemorrhagic Escherichia coli,EHEC）O157：H7菌株进行分型。结果4起事件中Shigetta．sonnei的PFGE结果显示,同起事件的DNA条带几乎一致;2起EHEC O157：H7暴发事件中,同起事件的PFGE条纹几乎一致,而3株散发菌株的PFGE条纹各异;12株EHEC O157：H7噬菌体分型结果也显示了比较好的分型力。结论PFGE具有分型能力强、重复性好等特点,能直观地判断肠道致病菌的亲缘关系,及时查明暴发流行的传染源,从而有效控制疫情的蔓延。     

Isolation and characterization of enterohaemorrhagic Escherichia coli O157:1H7 from cattle in Belgium and Poland

EHEC O157 were isolated from faeces of Belgian and Polish beef slaughter cattle. In Belgium, 1281 faecal samples were analysed by immunomagnetic separation [IMS] after enrichment in buffered peptone water from June 1998 till July 1999. Eighty-one samples (6.3%) were positive for E. coli O157. Phage type 8 was most frequently found. Bulls between 1 and 2 years old, slaughtered in September and October were most frequently found positive. Atypical biochemical features were observed in some isolates: 22 (27%) isolates were urease positive and 1 (1.2%) isolate was unable to ferment lactose. In Poland, 551 faecal samples, taken from January 1999 till December 1999, were examined using exactly the same techniques. Four faecal samples (0.7%) were positive for O157 EHEC, yielding seven phage type 8 isolates. All positive samples were from cattle younger than 2 years. Positive samples occurred in August, September and October.     

Sorbitol-fermenting, β-glucuronidase-positive, Shiga toxin-negative Escherichia coli O157:H7 in free-ranging red deer in South-Central Spain

We investigated the prevalence of Escherichia coli O157:H7 in free-ranging red deer in south-central Spain, to assess their potential as reservoir hosts of sorbitol-fermenting (SF) E. coli O157:H7 strains, which are emerging causes of hemolytic uremic syndrome in Europe. Fecal samples from 264 hunter-harvested Iberian red deer (Cervus elaphus) were collected in 25 different game estates and examined for E. coli O157:H7 by culture and PCR. E. coli O157:H7 was detected and isolated in 4 of the 25 game estates sampled (16%) and the isolates obtained (four in total) were further phenogenotypically characterized. One of them was biochemically typical of E. coli O157:H7, that is, neither fermented sorbitol nor exhibited β-glucuronidase (GUD) activity, and carried genes encoding Shiga toxins (Stx) 1 and 2, the intimin subtype γ1, the enterohemorrhagic E. coli (EHEC)-hemolysin, and the ter gene cluster. The rest of the isolates (three of four) fermented sorbitol, exhibited GUD activity after 18-24?h incubation, and carried genes encoding the intimin subtype γ1 and the EHEC-hemolysin, although no Stx-encoding genes were detected. All these atypical isolates carried the sfp gene cluster, lacked the ter gene cluster, and were unable to grow on cefixime tellurite sorbitol MacConkey agar, which are typical features of SF E. coli O157:H7 strains isolated from patients. In total, SF, GUD-positive, Stx-negative E. coli O157:H7 strains were isolated in 3 of the 25 game estates sampled (12%), with an overall sample-level prevalence of 1.1% (3/264). Our findings indicate that free-ranging red deer may be one of the possible reservoir hosts of Stx-negative derivatives of SF E. coli O157:H7.     

Prevalence of the eaeA gene in verotoxigenic Escherichia coli strains from dairy cattle in Southwest Ontario.

This study determined the prevalence of the eaeA gene and its relationship to serotype and type of verotoxin produced in a collection of 432 verotoxigenic Escherichia coli (VTEC) obtained from the faeces of healthy cows and calves in a systematic random survey involving 80 dairy farms in Southwest Ontario. A PCR amplification procedure involving primer pairs which target the conserved central region of the O157:H7 eaeA gene showed that 151 (35.2%) strains were positive for the eaeA gene. All isolates (9-21 for each O group) of O groups 5, 26, 69, 84, 103, 111, 145 and 157 were positive, whereas all isolates (7-34 for each O group) of O groups 113, 132, and 153 and serotype O156:NM (38 isolates) were negative for eaeA. Seventy-three percent of 130 isolates of eaeA-positive serotypes produced VT1 only compared with 20% of 253 isolates of eaeA-negative serotypes. We conclude that there is a strong association between certain O groups and the eaeA gene, that serotypes of eaeA-positive and eaeA-negative VTEC implicated in human and cattle disease are present at high frequency in the faeces of healthy cattle, that VT1 is more frequently associated with eaeA-positive than with eaeA-negative serogroups, and that the eaeA gene is more frequently found in VTEC from calves compared with VTEC from adult cattle.     

多重PCR快速检测食品中肠出血性大肠杆菌

本研究建立了一种快速检测食品中肠出血性大肠杆菌 (EHEC)的多重PCR方法。将样品增菌后 ,采用快速裂解吸附法制备模板 ,用多重PCR同时检测EHEC的三种毒力基因eaeA、hlyAB、slt1 2 ,相应扩增片段依次为 110 9、30 2、2 2 8bp。检测了 6 0株大肠杆菌和其它菌种。结果 12株大肠杆菌O15 7∶H7、1株大肠杆菌O2 6∶H11和 1株大肠杆菌O111∶H8同时检出上述三种基因 ,2株EAEC检出了eaeA基因 ,1株VTEC检出了slt1 2基因 ,其余 43株大肠杆菌和非大肠杆菌未检出上述基因。检测食品中EHEC时 ,从样品增菌培养到整个检测过程结束不超过 8小时 ,方法的检出限≤ 1 6cfu g(ml)。检测的 12 6份食品样品中 ,3份检出了EHEC(包括牛肉、猪肉和生菜各 1份 )。实验结果表明该法是一种特异性强、敏感性高、省时、省力的EHEC检测方法。     

不同来源产志贺毒素大肠埃希菌分布特征

目的探讨不同标本中产志贺样毒素大肠埃希菌（STEC）的分布特征。方法采集动物粪便、肉类食品和排污口污泥样品，常规分离大肠埃希菌，血清学分型，PCR鉴定产志贺样毒素（stx1，stx2）菌株。结果293份标本中鉴定出8株STEC，1株为产志贺毒素O157：H7型，2株为不产志贺毒素O157：H7型，5株为产志贺毒素非O157：H7型。结论STEC存在于不同来源的标本中，菌株表型与毒力因子存在一定差异。     

Identification of intermediate in evolutionary model of enterohemorrhagic Escherichia coli O157

Highly pathogenic enterohemorrhagic Escherichia coli (EHEC) O157 cause a spectrum of clinical signs that include diarrhea, bloody diarrhea, and hemolytic uremic syndrome. The current evolutionary model of EHEC O157:H7/H(-) consists of a stepwise evolution scenario proceeding from O55:H7 to a node (hypothetical intermediate) that then branches into sorbitol-fermenting (SF) O157:H(-) and non-SF (NSF) O157:H7. To identify this hypothetical intermediate, we performed single nucleotide polymorphism analysis by sequencing of 92 randomly distributed backbone genomic regions of 40 O157:H7/H(-) isolates. Overall, 111 single nucleotide polymorphisms were identified in 75/92 partial open reading frames after sequencing 51,041 nt/strain. The EHEC O157:H7 strain LSU-61 from deer occupied an intermediate position between O55:H7 and both O157 branches (SF and NSF O157), complementing the stepwise evolutionary model of EHEC O157:H7/H(-). The animal origin of this intermediate emphasizes the value of nonhuman reservoirs in the clarification of the evolution of human pathogens.     

Properties of Vero cytotoxin-producing Escherichia coli of human and animal origin belonging to serotypes other than O157:H7

Eight non-O157:H7 Vero cytotoxin (VT)-producing Escherichia coli (VTEC) strains isolated from ill persons and nine bovine and lamb strains of serogroups matching the human strains, were characterized for various properties known to be associated with E. coli virulence. Five different serogroups were represented: O5, O55, O103, O111 and O153. The bovine and lamb strains produced VT1, while 3 human strains produced VT1, 3 produced VT2 and 2 were positive for both VT1 and VT2. The strains were non-haemolytic on horse blood agar, did not produce either heat stable toxin A (STA) or heat labile toxin (LT), and were noninvasive. The CVD419 probe which has been proposed to identify enterohaemorrhagic E. coli (EHEC) hybridized with all of the O5 and O103 strains, none of the O55 and O153 strains, and 3 of the 4 O111 strains. The strains carried several different sized plasmids and hybridization of Southern blots with the CVD419 probe identified plasmids ranging in size from 42 x 10(6) to 90 x 10(6). The strains did not hybridize with the enteroadherence factor (EAF) probe derived from an enteropathogenic strain and associated with the ability to give localized adherence to HEp-2 cells. Nevertheless five of the strains adhered in a localized pattern to HEp-2 cells and Intestine 407 cells. Adhesion to either HEp-2 or Intestine 407 cells did not correlate with hybridization with the CVD419 probe or haemagglutinating properties.     

Enteroaggregative E. coli O104 from an outbreak of HUS in Germany 2011, could it happen again?

Enterohaemorrhagic E. coli (EHEC) particularly O157:H7 (Sequence type 11 complex), is the best documented and most well-known of E. coli that cause diarrhoea. The importance of EHEC lies in the severity of disease. Outbreaks can infect thousands of people causing bloody diarrhoea and haemolytic uremic syndrome (HUS) that in turn can result in protracted illness or even death. The ability of EHEC to colonise the human gut is normally associated with the presence of genes from another group of diarrhoeagenic E. coli, the enteropathogenic E. coli (EPEC), via the locus of enterocyte effacement. However, the massive outbreak in Germany was caused by an EHEC which had acquired virulence genes from yet another group of diarrhoeagenic E. coli, the enteroaggregative E. coli (EAEC). In reality EAEC is probably the most common bacterial cause of diarrhoea but is not identified in most diagnostic laboratories. This outbreak emphasises the importance of being able to detect all diarrhoeagenic E. coli and not to focus on E. coli O157:H7 alone. Routine surveillance systems for EAEC, a once ignored global pathogen, would go a long way to reaching this goal. This review describes methods for identifying non-O157 EHEC and describes the key genetic features of EHEC and EAEC. Our aim is to provide information for laboratories and policy makers which enables them to make informed decisions about the best methods available for detecting newly emergent strains of diarrhoeagenic E. coli.     

An outbreak of gastroenteritis in Osaka,Japan due to Escherichia coli serogroup O166:H15 that had a coding gene for enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1)

In an outbreak of gastroenteritis on 23 July 1996, in Osaka, Japan, 54 of 91 persons who had attended a meeting the previous day became ill. Escherichia coli O166:H15 was isolated from stool specimens of patients (29/33, 88%). Laboratory tests for other bacterial pathogens and viruses were negative. The E. coli 0166 organisms did not adhere to HEp-2 cells in a localized, diffuse, or enteroaggregative manner. The organisms did not express known enterotoxigenic E. coli (ETEC) colonization factors. In polymerase chain reaction tests, the bacteria did not have coding genes for shigatoxin of enterohemorrhagic E. coli (EHEC), heat-labile, or heat-stable enterotoxin of ETEC, attachment and effacement (eaeA) of EPEC, or invasion (invE) of enteroinvasive E. coli (EIEC). Consequently, they could not be assigned to any of the recognized diarrhoeagenic groups of E. coli: EPEC, ETEC, EHEC, EIEC, enteroaggregative E. coli (EAggEC), or diffusely adhering E. coli. However, the organisms possessed the EAggEC heat-stable enterotoxin (EAST1) gene. To our knowledge, this is the first report of an outbreak caused by E. coli that did not have well-characterized virulence genes other than EAST1. The isolates showed the same DNA banding pattern in pulsed-field gel electrophoresis after digestion with the restriction enzymes XbaI or NotI. Three O166:H15 strains isolated from two sporadic cases and another outbreak during 1997-8 were distinct, indicating that multiple clones have spread already. We propose that diarrhoeal specimens should be examined for E. coli possessing the EAST1 gene.