Reference laboratories

European Union Reference Laboratory for Escherichia coli

Shiga toxin-producing Escherichia coli (STEC)

STEC infections constitute a major public health concern, as they are able to cause severe illnesses such as haemorrhagic colitis and haemolytic uremic syndrome, especially among children and the elderly. STEC are zoonotic pathogens, with ruminants being recognized as the major natural reservoir. The large number of outbreaks occurring all over the world underlines the importance of these pathogens and highlights the need for both mandatory disease notification and cooperation between laboratories within and beyond state boundaries. The majority of the severe cases worldwide are caused by strains belonging to serogroups O157, O26, O111, O103 and O145, but, as the detection and typing technology improves, other serogroups are constantly reported but the pathogenicity assessment increasingly relies more on the determination of the virulence genes asset than on the phenotypic markers as the serogroup [1]. Recent findings on the pathogenesis of STEC infection and some emerging modes of transmission are summarised in the following sections.

About STEC

Escherichia coli is part of the normal microflora of the gastrointestinal tract of mammals and birds, but certain strains have been associated with gastrointestinal diseases in both humans and animals. These E. coli strains have been categorised into pathogenicity groups (pathogroups), based on their virulence properties [2]. One of these groups is characterised by the production of potent cytotoxins that inhibit the protein synthesis within eukaryotic cells. These toxins, previously termed verocytotoxins (VT) because of their activity on Vero cells, are now referred to as Shiga toxins (Stx), because of their similarity with the toxin produced by Shigella dysenteriae [3]. Therefore, these strains are termed Stx-producing E. coli (STEC).

Certain STEC have been firmly associated with bloody diarrhoea and haemolytic uraemic syndrome (HUS) in industrialised countries [4, 2]. The majority of the cases of severe disease are caused by strains of serotype O157:H7, but infections sustained by strains belonging to serogoups other than O157, like O26, O111, O103, and O145 have been increasingly reported [2, 5]. These strains are usually referred to as non–O157 STEC.

Virulence factors

Shiga toxins

Stxs are considered to be the major virulence factor of STEC and comprise a family of structurally related cytotoxins with similar biological activity. The two main groups consist of Stx1, which is nearly identical to the toxin of S. dysenteriae type 1, and Stx2, which shares less than 60 % amino acid sequence with Stx1 [3]. The genetic information for the production of Stx1 and Stx2 is located in the genome of lambdoid prophages integrated in the STEC chromosome [3]. Whereas Stx1 shows only little sequence variations, several subtypes and variants of Stx2 with altered sequence, antigenic or biological characteristics have been described. Epidemiological studies have revealed that Stx2 is more associated with severe human disease than Stx1 [6]. A certain number of subtypes are produced by strains of animal origin and are rarely observed in human isolates: Stx2e is mainly found in STEC causing oedema disease in pigs and Stx2f appears to be closely associated with STEC of avian origin [7], although the latter has been described in STEC isolated from human HUS cases [8].

Attaching and effacing adhesion

Most STEC causing the severe disease, such as HUS and HC, colonise the intestinal mucosa with a mechanism that subverts the epithelial cell function [9] and induce a characteristic histopathologic lesion, defined as "attaching and effacing"(A/E). The A/E lesion is due to marked cytoskeletal changes and is characterised by effacement of microvilli and intimate adherence between the bacteria and the epithelial cell membrane, with accumulation of polymerised actin directly beneath the adherent bacteria [2].

The complex mechanism of A/E adhesion is genetically governed by a large pathogenicity island (PAI) defined as Locus of Enterocyte Effacement (LEE) [2, 9]. 

Other virulence factors

Genetic analysis of the complete DNA sequence of STEC O157:H7 [10] showed that almost 20 % of its chromosome is constituted by foreign DNA not present in the chromosome of E. coli K-12 and that has been probably acquired from other bacterial species through horizontal gene transfer. Similarly to the LEE, other regions of this foreign DNA can be considered as putative PAIs since they carry virulence-associated genes, show a lower GC content, and are inserted in tRNA loci. In particular, a PAI termed O#122 is present in most EHEC and enteropathogenic E. coli (EPEC), but not in other groups of E. coli [11]. 

STEC O157 possess a large virulence plasmid of approximately 90 Kb termed pO157. The nucleotide sequence of this plasmid showed that it encodes 35 proteins, some of which are presumably involved in the pathogenesis of EHEC infections [12]. The enterohaemolysin (hly) operon is considered the best marker of the presence of pO157 and is also present in the large plasmids that can be detected in most non-O157 EHEC strains [13] Other putative virulence factors harboured by this plasmid comprise a katalase-peroxyidase and a serine protease, encoded by katP and espP genes, respectively [14]. Another virulence gene, termed toxB, has been recently described in pO157 [15] and it appears to be present in all the STEC O157 isolates [16]. 

STEC are zoonotic pathogens

STEC can be found in the gut of numerous animal species, but ruminants have been identified as a major reservoir of STEC that are highly virulent to humans, in particular STEC O157. 

Cattle are considered to be the most important source of human infections with STEC O157, being asymptomatic eliminators of the organism, which is a transient member of their normal gut micro flora [4]. STEC have also been frequently isolated from the intestinal content of sheep that is now considered a reservoir for human infection [17]. STEC has also been isolated from goats [18] and water buffalo [19].

STEC have been sporadically isolated from mammals other than ruminants, like pigs [20], horses [21], dogs [22] and farmed rabbits [23] but these species are not considered as actual hosts but rather as vectors transiently colonised after a contact with ruminant dejections. 

Epidemiology of STEC infections 

During the 1980s, most of the outbreaks of STEC O157 infection were food-borne and the food vehicles implicated were mostly inadequately cooked beef products and unpasteurised milk [2]. In the past twenty years, several outbreaks have been associated with low pH products like fermented salami, mayonnaise and yogurt [24]. This has highlighted the tolerance of E. coli O157 to acidic pH and its ability to survive the processes of fermentation and drying. In addition, waterborne outbreaks and outbreaks associated with other types of environment-related exposures have been increasingly reported [25, 26]. The dispersion of untreated manure in the environment can cause the contamination of different items, which can then act as secondary vehicles of human infections [4, 25, 27].

An increasing spectrum of fruits and vegetables grown on fields fertilised with ruminants’ manure or contaminated during harvesting or processing has been involved in outbreaks [4, 25, 26]. 

Control strategies

Food products of ruminant origins that are free from STEC are not feasible in practice. However, their occurrence can be minimised by applying high standards of hygiene in all the steps of the food production chain.

At the farm level, good hygiene and management practices remains at the present the best way to reduce the spread and persistence of STEC in the farm. 

STEC can survive in bovine faeces for a considerable time [25], therefore the handling of the animal excreta represents an important issue and manure and slurries should be properly composted to ensure the reduction of the microbial load [28, 29].

Farmers and people visiting farms should apply hygiene practices. In particular, farms receiving school visits must ensure that adults always control children, facilities for hand washing are easily available, and areas for food consume are clearly separated from those where the animals are kept. 

At the abattoir level, good hygiene and manufacturing practices as well as implementation of HACCP will contribute in reducing faecal contamination of carcasses. 

The general principles of food hygiene will be effective in preventing STEC infections also at the processing and retail levels of the food chain. 

Hybrid or cross-pathotype STEC 

The plasticity of E. coli genome and the localization of the majority of the virulence genes on mobile genetic elements imply that pathogenic clones displaying hybrid repertoires of virulence features, termed hybrid or cross-pathotype strains, may emerge. As a matter of fact, the ability to produce Shiga toxin has been reported in strains also displaying virulence genes typical of other pathotypes, including Enteroaggregative E. coli (EAEC) and Enterotoxigenic E. coli (ETEC) [30, 31]. In particular, the outbreak of severe infections caused by an O104:H4 EAEC-STEC across Europe in 2011 was the largest ever reported for a STEC and highlighted the importance of the emergence of such hybrid pathogenic clones [32]. It was postulated that in STEC strains lacking the LEE locus the expression of other colonization mechanisms, such as that typical of Enteroaggregative E. coli strains, could confer high pathogenicity. More recently, Stx-producing ETEC (ETEC-STEC) were shown to circulate in animals and were also reported as causative agents of human disease [31] and associated with a case of severe human infection [33]. Interestingly, the strain responsible of the latter case lacked the LEE locus and was shown to harbour a hybrid plasmid carrying STEC-associated virulence genes, such as hlyA, and ETEC virulence genes, encoding porcine variant of the Heat Stable enterotoxin STp and a novel variant of the F4 ETEC fimbriae.

An additional emerging group of E. coli showing hybrid virulence features is represented by STEC harbouring virulence genes associated with ExPEC. In detail, ExPEC-STEC strains belonging to O80:H2 serotype have been associated not only with clinical features of STEC, but also with bacteriaemia [34]. These strains have been described to harbour a mosaic plasmid (pR444_A), which combines extraintestinal virulence genes including hlyF, ompT and iroBCDEN, with genes conferring resistance to multiple antimicrobials [35]. Additional strains of various setotypes belonging to the same Sequence Type (ST) 301 of O80:H2 ExPEC-STEC were described to harbour a similar asset of virulence genes. Such STEC showing ExPEC virulence genes were isolated either in cases of human infection or in animal sources [36].




[1] Koutsoumanis K., Allende A., Alvarez-Ordonez A., Bover-Cid S., Chemaly M., Davies R., De Cesare A., Herman L., Hilbert F., Lindqvist R., Nauta M., Peixe L., Ru G., Simmons M., Skandamis P., Suffredini E., Jenkins C., Monteiro Pires S., Morabito S., Niskanen T., Scheutz F., da Silva Felicio M.T., Messens W., Bolton D., Pathogenicity assessment of Shiga toxin-producing Escherichia coli (STEC) and the public health risk posed by contamination of food with STEC, EFSA BIOHAZ Panel, SCIENTIFIC OPINION ADOPTED: 11 December 2019, doi: 10.2903/j.efsa.2020.5967.

[2] Nataro J.P. Kaper J.B., Diarrheagenic Escherichia coli, Clin. Microbiol. Rev. 11 (1998) 142-201. 

[3] Melton-Chelsa A.R., O'Brien A., Structure, biology, and relative toxicity of Shiga toxin family members for cells and animals. In Kaper J.B., O’Brien A. D. (Eds.), Escherichia coli O157:H7 and other Shiga toxin-producing E. coli strains. American Society for Microbiology, Washington, DC. (1998) 121-128.

[4] Caprioli A., Morabito S., Brugere H, Oswald E., Enterohaemorrhagic Escherichia coli: emerging issues on virulence and modes of transmission, Vet. Res. 36 (2005) 289311.

[5] Tozzi A.E., Caprioli A., Minelli F., Gianviti A., De Petris L., Edefonti A., Montini G., Ferretti A., De Palo T., Gaido M., Rizzoni G., Hemolytic Uremic Syndrome Study Group, Shiga toxinproducing Escherichia coli infections associated with hemolytic uremic syndrome, Italy, 1988-2000, Emerg. Infect. Dis. 9 (2003) 106-108.

[6] Boerlin P., McEwen S.A., Boerlin-Petzold F., Wilson J.B., Johnson R.P., Gyles C.L., Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans, J. Clin. Microbiol. 37 (1999) 497-503.

[7] Schmidt H., Scheef J., Morabito S., Caprioli A., Wieler L.H., Karch H., A new Shiga toxin 2 variant (Stx2f) from Escherichia coli isolated from pigeons, Appl. Environ. Microbiol. 66 (2000) 12051208.

[8] Grande L., Michelacci V., Bondì R., Gigliucci F., Franz E., Askari Badouei M., Schlager S., Minelli F., Tozzoli R., Caprioli A., Morabito S., Whole-Genome Characterization and Strain Comparison of VT2f-Producing Escherichia coli Causing Hemolytic Uremic Syndrome, Emerg Infect Dis. (2016);22(12):2078-2086. doi: 10.3201/eid2212.160017. Epub 2016 Dec 15.

[9] Frankel G., Phillips A. D., Rosenshine I., Dougan G., Kaper J. B. Knutton S., Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements, Mol. Microbiol. 30 (1998) 911-921.

[10] Perna N.T., Plunkett G. 3rd, Burland V., Mau B., Glasner J.D., Rose D.J., Mayhew G. F., Evans P.S., Gregor J., Kirkpatrick H.A., Posfai G., Hackett J., Klink S., Boutin A., Shao Y., Miller L., Grotbeck E.J., Davis N.W., Lim A., Dimalanta E.T., Potamousis K.D., Apodaca J., Anantharaman T.S., Lin J., Yen G., Schwartz D.C., Welch R.A. Blattner F.R., Genome sequence of enterohaemorrhagic Escherichia coli O157:H7, Nature 409 (2001) 529-533. 

[11] Morabito S., Tozzoli R., Oswald E., Caprioli A., A mosaic pathogenicity island made up of the locus of enterocyte effacement and a pathogenicity island of Escherichia coli O157:H7 is frequently present in attaching and effacing E. coli, Infect. Immun. 71 (2003) 3343-3348.

[12] Burland V., Shao Y., Perna N.T., Plunkett G., Sofia H.J., Blattner F.R., The complete DNA sequence and analysis of the large virulence plasmid of Escherichia coli O157:H7, Nucleic Acids Res. 26 (1998) 4196-4204. 

[13] Brunder W., Schmidt H., Frosch M., Karch H., The large plasmids of Shiga-toxin-producing Escherichia coli (STEC) are highly variable genetic elements, Microbiology 145 (1999)1005-1014.

[14] Schmidt H., Bitzan M., Karch H., Pathogenic aspects of Shiga toxin-producing E. coli infections in humans. In Duffy G., Garvey P., McDowell D. (Eds.), Verocytotoxigenic Escherichia coli, Food & Nutrition Press INC. (2001) 241-262.

[15] Tatsuno I., Kimura H., Okutani A., Kanamaru K., Abe H., Nagai S., Makino K., Shinagawa H., Yoshida M., Sato K., Nakamoto J., Tobe T. Sasakawa C., Isolation and characterization of miniTn5Km2 insertion mutants of enterohemorragic Escherichia coli O157:H7 deficient in adherence to Caco-2 cells, Infect. Immun. 68 (2000) 5943-5952. 

[16] Tozzoli R., Caprioli A., Morabito S., Detection of toxB, a plasmid virulence gene of Escherichia coli O157, in enterohemorrhagic and enteropathogenic E. coli, J. Clin. Microbiol. 43(2005) 40524056.

[17] Heuvelink A.E., Van den Biggelaar F.L., De Boer E., Herbes R.G., Melchers W.J., Huis in 't Veld J.H., Monnens L.A., Isolation and characterization of verocytotoxin-producing Escherichia coli O157 strains from Dutch cattle and sheep, J. Clin. Microbiol. 36 (1998) 878-882.

[18] Pritchard G.C., Willshaw G.A., Bailey J.R.,Carson T., Cheasty T., Verocytotoxin-producing Escherichia coli O157 on a farm open to the public: outbreak investigation and longitudinal bacteriological study, Vet. Rec. 147 (2000) 259-264. 

[19] Galiero G., Conedera G., Alfano D., Caprioli A., Isolation of verocytotoxin-producing Escherichia coli O157 from water buffaloes (Bubalus bubalis) in southern Italy, Vet Rec. 156 (2005) 382-383.

[20] Bonardi S., Brindani F., Pizzin G., Lucidi L., D'Incau M., Liebana E., Morabito S., Detection of Salmonella spp., Yersinia enterocolitica and verocytotoxin-producing Escherichia coli O157 in pigs at slaughter in Italy, Int. J. Food. Microbiol. 85 (2003) 101-110. 

[21] Chalmers R.M., Salmon R.L., Willshaw G.A., Cheasty T., Looker N., Davies I., Wray C., Vero-cytotoxin-producing Escherichia coli O157 in a farmer handling horses, Lancet 349 (1997) 1816.

[22] Trevena W.B., Hooper R.S., Wray C., Willswaw G.A., Cheasty T., Domingue G., Vero cytotoxin-producing Escherichia coli O157 associated with companion animals, Vet. Rec. 138 (1996) 400. 

[23] Leclercq A., Mahillon J., Farmed rabbits and ducks as vectors for VTEC O157:H7, Vet. Rec. 152 (2003) 723-724.

[24] Meng J., Doyle M.P., Microbiology of Shiga-Toxin-Producing Escherichia coli in foods, In: Kaper, J.B., O’Brien, A. D. (Eds.), Escherichia coli O157:H7 and other Shiga-Toxin-Producing E. coli. American Society for Microbiology, Washington, DC, (1998) 92-108.

[25] McDowell D.A., Sheridan J.J., Survival and growth of Vero cytotoxin-producing E. coli in the environment, In Duffy G., Garvey P., McDowell D. (Eds.), Verocytotoxigenic Escherichia coli, Food & Nutrition Press INC. (2001) 279-304. 

[26] Tozzi A.E., Gorietti S., Caprioli A., Epidemiology of human infections by Escherichia coli O157 and other verocytotoxinproducing E .coli, In Duffy G., Garvey P., McDowell D. (Eds.), Verocytotoxigenic Escherichia coli, Food & Nutrition Press INC. (2001) 161-179.

[27] Coia J.E., Sharp J.C., Campbell D.M., Curnow J., Ramsay C.N., Environmental risk factors for sporadic Escherichia coli O157 infection in Scotland: results of a descriptive epidemiology study, J. Infect. 36 (1998) 317-321. 

[28] Jiang X., Morgan J., Doyle M.P., Fate of Escherichia coli O157:H7 during composting of bovine manure in a laboratoryscale bioreactor, J. Food Prot. 66 (2003) 25-30.

[29] Lung A.J., Lin C.M., Kim J.M., Marshall M.R., Nordstedt R., Thompson N.P., Wei C.I., Destruction of Escherichia coli O157:H7 and Salmonella enteritidis in cow manure composting, J. Food Prot. 64 (2001) 1309-1314.

[30] Scheutz F., Møller Nielsen E., Frimodt-Møller J., Boisen N., Morabito S., Tozzoli R., Nataro J.P., Caprioli A., 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(24):19889. doi: 10.2807/ese.16.24.19889-en.

[31] Nyholm O., Heinikainen S., Pelkonen S., Hallanvuo S., Haukka K., Siitonen A., Hybrids of Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Among Human and Animal Isolates in Finland, Zoonoses Public Health. (2015);62(7):518-24. doi: 10.1111/zph.12177. Epub 2015 Jan 9.

[32] Frank C., Werber D., Cramer J.P., Askar M., Faber M., an der Heiden M., Bernard H., Fruth A., Prager R., Spode A., Wadl M., Zoufaly A., Jordan S., Kemper M.J., Follin P., Müller L., King L.A., Rosner B., Buchholz U., Stark K., Krause G., HUS Investigation Team, Epidemic profile of Shiga-toxin-producing Escherichia coli O104:H4 outbreak in Germany Collaborators, Affiliations expand, N Engl J Med. (2011);365(19):1771-80. doi: 10.1056/NEJMoa1106483.

[33] Michelacci V., Maugliani A., Tozzoli R., Corteselli G., Chiani P., Minelli F., Gigliucci F., Arancia S., Conedera G., Targhetta C., Pierasco A., Collini L., Parisi A., Scavia G., Morabito S., Characterization of a novel plasmid encoding F4-like fimbriae present in a Shiga-toxin producing enterotoxigenic Escherichia coli isolated during the investigation on a case of hemolytic-uremic syndrome, Int J Med Microbiol. (2018);308(7):947-955. doi: 10.1016/j.ijmm.2018.07.002. Epub 2018 Jul 11.

[34] Mariani-Kurkdjian P., Lemaître C., Bidet P., Perez D., Boggini L., Kwon T., Bonacorsi S., Haemolytic-uraemic syndrome with bacteraemia caused by a new hybrid Escherichia coli pathotype, New Microbes New Infect. (2014);2(4):127-31. doi: 10.1002/nmi2.49. Epub 2014 May 27.

[35] Cointe A., Birgy A., Mariani-Kurkdjian P., Liguori S., Courroux C., Blanco J., Delannoy S., Fach P., Loukiadis E., Bidet P., Bonacorsi S., Emerging Multidrug-Resistant Hybrid Pathotype Shiga Toxin-Producing Escherichia coli O80 and Related Strains of Clonal Complex 165, Europe, Emerg Infect Dis. (2018);24(12):2262-2269. doi: 10.3201/eid2412.180272.

[36] Gigliucci F., van Hoek A., Chiani P., Knijn A., Minelli F., Scavia G., Franz E., Morabito S., Michelacci V., Genomic Characterization of hlyF-positive Shiga Toxin-Producing Escherichia coli, Italy and the Netherlands, 2000-2019, Emerg Infect Dis. (2021);27(3):853-861. doi: 10.3201/eid2703.203110.