Diarrheagenic Escherichia coli

Tânia A.T. Gomes Waldir P. Elias Isabel C.A. Scaletsky Beatriz E.C. Guth Juliana F. Rodrigues Roxane M.F. Piazza Luís C.S. Ferreira Marina B. Martinez About the authors

ABSTRACT

Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes.

Keywords:
Escherichia coli; Diarrhea; Pathogenic mechanisms; Virulence factor; Epidemiology

The genus Escherichia, which was named after the German pediatrician Theodor Escherich, consists of facultative anaerobic Gram-negative bacilli that belong to the family Enterobacteriaceae.11 Ewing WH. Edwards and Ewing's Identification of Enterobacteriaceae. 4th ed. New York: Elsevier; 1986. The genus type species Escherichia coli is widely distributed, where it is the major facultative anaerobe inhabiting the large intestine of humans and warm-blooded animals.22 Conway PL. Microbial ecology of the human large intestine. In: Gibson GR, Macfarlane GT, eds. Human Colonic Bacteria: Role in Nutrition, Physiology and Pathology. Boca Raton, FL, USA: CRC Press; 1995:1-24. Although most E. coli strains live harmlessly in the colon and seldom cause disease in healthy individuals, a number of pathogenic strains can cause intestinal and extraintestinal diseases both in healthy and immunocompromised individuals.33 Kaper JB, Nataro JP, Mobley HLT. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004;2(2):123-140.

Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children.44 World Health Organization. World Health Statistics. Geneva, Switzerland: WHO Press; 2012. Low- and middle-income countries in Africa, Asia and Latin America are the most affected regions with diarrheal diseases occurring more often with lethal outcomes mainly due to poor living conditions (inadequate water supplies, poor environmental hygiene and sanitation, and insufficient education).55 Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26(4):822-880.

E. coli strains involved in diarrheal diseases are one of the most important of the various etiological agents of diarrhea, where strains have evolved by the acquisition, through horizontal gene transfer, of a particular set of characteristics that have successfully persisted in the host.33 Kaper JB, Nataro JP, Mobley HLT. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004;2(2):123-140.,55 Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26(4):822-880.,66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli (DEC).66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201. The DEC pathotypes differ regarding their preferential host colonization sites, virulence mechanisms, and the ensuing clinical symptoms and consequences, and are classified as enteropathogenic E. coli (EPEC), enterohemorrhagic (Shiga toxin-producing) E. coli (EHEC/STEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC).

Each of these pathotypes represents a group of clones that share specific virulence factors. Nevertheless, it should be pointed out that the plasticity of the E. coli genome has hindered the identification of certain E. coli isolates as a pathotype, because some isolates combine the main virulence characteristics of different pathotypes and are thus considered potentially more virulent hybrid pathogenic strains.55 Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26(4):822-880.

Another less well-defined pathotype has been described, that is, the diffusely-adherent E. coli (DAEC) pathotype, which comprises strains that adhere to epithelial cells in a diffused distribution.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201. Despite their classification as a group distinct from the other pathotypes, the designation of DAEC as a different DEC pathotype requires further epidemiological studies, which have been hampered by the difficulties in its identification and classification.55 Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26(4):822-880. Furthermore, certain E. coli strains that have been classified as the adherent invasive E. coli (AIEC) pathotype, comprise one of the potential agents for Crohn's disease (CD). CD is an inflammatory bowel disease (IBD), which is thought to be caused by a combination of factors (genetics, the intestinal microbiota, environmental factors, and enteric pathogens).77 Rolhion N, Darfeuille-Michaud A. Adherent-invasive Escherichia coli in inflammatory bowel disease. Inflamm Bowel Dis. 2007;13(10):1277-1283.,88 Cieza RJ, Cao AT, Cong Y, Torres AG. Immunomodulation for gastrointestinal infections. Expert Rev Anti Infect Ther. 2012;10(3):391-400.

Diarrheal episodes due to DEC infections are an important public health issue among children and adults in developing countries, because of their association with morbidity and mortality of children less than five years of age. It was our aim with this review to gather information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major DEC pathotypes with emphasis on the studies conducted in Brazil.

Typical and atypical enteropathogenic E. coli

The term enteropathogenic E. coli (EPEC) was first used in 1995 by Neter et al.,99 Neter E, Westphal O, Luderitz O, Gino RM, Gorzynski EA. Demonstration of antibodies against enteropathogenic Escherichia coli in sera of children of various ages. Pediatrics. 1995;16:801-807. to describe a number of E. coli strains epidemiologically related to a series of outbreaks of infantile diarrhea in the 1940s and 1950s.1010 Bray J. Isolation of antigenically homogeneous strains of Bacterium coli neopolitanum from summer diarrhoea of infants. J Pathol Bacteriol. 1945;57(2):239-247.,1111 Robins-Browne RM. Traditional enteropathogenic Escherichia coli of infantile diarrhea. Rev Infect Dis. 1987;9(1):28-53. Originally identified by serotype, EPEC are now defined as those E. coli strains having the ability to cause diarrhea, to produce a histopathology on the intestinal epithelium known as the attaching and effacing (AE) lesion, and the inability to produce Shiga toxins and heat-labile (LT) or heat-stable (ST) enterotoxins.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201.

Improvements in techniques allowing a better understanding of the genome and virulence mechanisms among EPEC strains over the years have led to the sub-classification of EPEC into typical EPEC (tEPEC) and atypical EPEC (aEPEC).33 Kaper JB, Nataro JP, Mobley HLT. Pathogenic Escherichia coli. Nat Rev Microbiol. 2004;2(2):123-140.,1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513. Typical EPEC strains causing human infectious diarrhea possess a large virulence plasmid known as the EPEC adherence factor (EAF) plasmid (pEAF), which encodes the type IV fimbriae called the bundle-forming pilus (BFP), while aEPEC do not possess this plasmid.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201.,1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513.

The majority of tEPEC strains fall into well-recognized O serotypes. Classical EPEC O serogroups include O55, O86, O111, O114, O119, O127, and O142. The most common H antigens associated with EPEC are the H6 and H2 antigens.1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513.

13 Ørskov F, Whittam TS, Cravioto A, Ørskov I. Clonal relationships among classic enteropathogenic Escherichia coli (EPEC) belong to different O groups. J Infect Dis. 1990;162(1):76-81.

14 Whittam TS, McGraw EA. Clonal analysis of EPEC serogroups. Rev Microbiol. 1996;27:7-16.
-1515 Gomes TAT, González-Pedrajo B. Enteropathogenic Escherichia coli (EPEC). In: Torres AG, ed. Pathogenic Escherichia coli in Latin America. Sharjah, United Arab Emirates: Betham Science Publishers Ltd.; 2010:66-126. A less common EPEC type is H34, and a number of tEPEC strains are classified as non-motile (H-) in conventional tests. Typical EPEC strains belonging to non-classical serotypes have also been reported.1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513.,1616 Gomes TAT, Griffin PM, Ivey C, Trabulsi LR, Ramos SRTS. EPEC infections in São Paulo. International Symposium on Enteropathogenic Escherichia coli (EPEC), São Paulo, SP. Rev Microbiol Soc Bras Microbiol. 1996;27:25-33.

Based on multilocus enzyme electrophoresis analysis (MLEE) of allelic differences between housekeeping genes, tEPEC strains have been subtyped into two major lineages, previously designated EPEC1 and EPEC2.1313 Ørskov F, Whittam TS, Cravioto A, Ørskov I. Clonal relationships among classic enteropathogenic Escherichia coli (EPEC) belong to different O groups. J Infect Dis. 1990;162(1):76-81.,1414 Whittam TS, McGraw EA. Clonal analysis of EPEC serogroups. Rev Microbiol. 1996;27:7-16. EPEC1 includes widespread serotypes such as O55:H6 and O119:H6, whereas EPEC2 consists of serotypes with more limited occurrence such as O111:H2 and O114:H2. Based on a whole-genome phylogeny and analysis of type III secretion system (T3SS) effectors, tEPEC strains have been demonstrated to cluster in three main lineages, designated EPEC1, EPEC2, and EPEC4, which probably acquired the locus of enterocyte effacement (LEE) region and pEAF independently.1717 Hazen TH, Sahl JW, Fraser CM, Donnenberg MS, Scheutz F, Rasko DA. Refining the pathovar paradigm via phylogenomics of the attaching and effacing Escherichia coli. PNAS. 2013;110(31):12810-12815.

In turn, aEPEC belong to a large diversity of classical and non-classical serotypes.1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513.,1616 Gomes TAT, Griffin PM, Ivey C, Trabulsi LR, Ramos SRTS. EPEC infections in São Paulo. International Symposium on Enteropathogenic Escherichia coli (EPEC), São Paulo, SP. Rev Microbiol Soc Bras Microbiol. 1996;27:25-33.,1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149. Over 20% of strains of non-classical EPEC serotypes are O non-typeable and the O-typeable strains belong to more than 4200 different serotypes, with many non-motile and H non-typeable strains.1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513.,1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149. Interestingly, it has been found that 35% of the aEPEC strains also belong to the tEPEC lineages.1717 Hazen TH, Sahl JW, Fraser CM, Donnenberg MS, Scheutz F, Rasko DA. Refining the pathovar paradigm via phylogenomics of the attaching and effacing Escherichia coli. PNAS. 2013;110(31):12810-12815. Thus, it has been hypothesized that at least some aEPEC may have originated from tEPEC strains that lost pEAF in the host or in the environment.1717 Hazen TH, Sahl JW, Fraser CM, Donnenberg MS, Scheutz F, Rasko DA. Refining the pathovar paradigm via phylogenomics of the attaching and effacing Escherichia coli. PNAS. 2013;110(31):12810-12815.,1919 Levine MM, Nataro JP, Karch H, et al. The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. J Infect Dis. 1985;152(3):550-559.,2020 Vieira MA, Andrade JR, Trabulsi LR, et al. Phenotypic and genotypic characteristics of Escherichia coli strains of non-enteropathogenic E. coli (EPEC) serogroups that carry eae and lack the EPEC adherence factor and Shiga toxin DNA probe sequences. J Infect Dis. 2001;183(5):762-772.

Virulence factors, mechanisms and pathogenesis

Typical EPEC strains adhere to HeLa, HEp-2, and other cell lines and to organ cultures in vitro in a distinctive pattern of three-dimensional microcolonies, a so-called localized adherence (LA) pattern.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201.,2121 Scaletsky IC, Silva ML, Trabulsi LR. Distinctive patterns of adherence of enteropathogenic Escherichia coli to HeLa cells. Infect Immun. 1984;45(2):534-536. A similar adherence pattern has been seen in tissue biopsies of EPEC-infected humans.2222 Rothbaum R, McAdams AJ, Giannella R, Partin JC. A clinicopathological study of enterocyte-adherent Escherichia coli: a cause of protracted diarrhea in infants. Gastroenterology. 1982;83(2):441-454.

The LA phenotype is mediated by the BFP,2323 Girón JA, Ho AS, Schoolnik GK. An inducible bundle-forming pilus of enteropathogenic Escherichia coli. Science. 1991;254(5032):710-713. which also contributes to antigenicity, autoaggregation, and biofilm formation.2323 Girón JA, Ho AS, Schoolnik GK. An inducible bundle-forming pilus of enteropathogenic Escherichia coli. Science. 1991;254(5032):710-713.

24 Bieber D, Ramer SW, Wu CY, et al. Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli. Science. 1998;280:2114-2118.

25 Vuopio-Varkila J, Schoolnik GK. Localized adherence by enteropathogenic Escherichia coli is an inducible phenotype associated with the expression of new outer membrane proteins. J Exp Med. 1991;174(5372):1167-1177.

26 Moreira CG, Palmer K, Whiteley M, et al. Bundle-forming pili and EspA are involved in biofilm formation by enteropathogenic Escherichia coli. J Bacteriol. 2006;188(11):3952-3961.
-2727 Hyland RM, Sun J, Griener TP, et al. The bundling pilin protein of enteropathogenic Escherichia coli is an N-acetyllactosamine-specific lectin. Cell Microbiol. 2008;10(1):177-187. An operon of 14 genes contained on the pEAF is necessary for BFP expression, with bfpA encoding the major structural subunit (bundlin)2828 Stone KD, Zhang H, Carlson LK, Donnenberg MS. A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus. Mol Microbiol. 1996;20(2):325-337. and being highly conserved among EPEC1 and EPEC2 strains.

The self-transmissible pEAF pMAR2 is found among strains of the EPEC1 lineage and contains an intact transfer region, unlike pB171, which is more common among EPEC2 strains.2929 Tobe T, Hayashi T, Han C, Schoolnik GK, Ohtsubo E, Sasakawa C. Complete DNA sequence and structural analysis of the enteropathogenic Escherichia coli adherence factor plasmid. Infect Immun. 1999;67(10):5455-5462.,3030 Brinkley C, Burland V, Keller R, et al. Nucleotide sequence analysis of the enteropathogenic Escherichia coli adherence factor plasmid pMAR7. Infect Immun. 2006;74(9):5408-5413. Besides the bfp gene cluster, encoding BFP,2323 Girón JA, Ho AS, Schoolnik GK. An inducible bundle-forming pilus of enteropathogenic Escherichia coli. Science. 1991;254(5032):710-713. the pEAF carries the per locus, encoding the transcriptional activator called plasmid-encoded regulator (Per).2929 Tobe T, Hayashi T, Han C, Schoolnik GK, Ohtsubo E, Sasakawa C. Complete DNA sequence and structural analysis of the enteropathogenic Escherichia coli adherence factor plasmid. Infect Immun. 1999;67(10):5455-5462. Between pMAR2 and pB171, the bfp and per loci share 99% sequence similarity,3030 Brinkley C, Burland V, Keller R, et al. Nucleotide sequence analysis of the enteropathogenic Escherichia coli adherence factor plasmid pMAR7. Infect Immun. 2006;74(9):5408-5413. and both BFP and PerA have been shown to contribute to virulence in human volunteers.2424 Bieber D, Ramer SW, Wu CY, et al. Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic Escherichia coli. Science. 1998;280:2114-2118. Recent comparative genomics of the EAF plasmids from diverse EPEC phylogenomic lineages demonstrated significant plasmid diversity even among isolates within the same phylogenomic lineage.3131 Hazen TH, Kaper JB, Nataro JP, Rasko DA. Comparative genomics provides insight into the diversity of the attaching and effacing Escherichia coli virulence plasmids. Infect Immun. 2015;83(10):4103-4117.

Typical EPEC have the ability to form tight, spherical, bacterial autoaggregates when grown in liquid culture.3232 Nougayrède JP, Fernandes PJ, Donnenberg MS. Adhesion of enteropathogenic Escherichia coli to host cells. Cell Microb. 2003;5(6):359-372. Like LA, autoaggregation requires BFP. Typical EPEC also form biofilms on abiotic surfaces under static conditions, or in a flow through continuous culture system, and a model of EPEC biofilm formation has been proposed.2626 Moreira CG, Palmer K, Whiteley M, et al. Bundle-forming pili and EspA are involved in biofilm formation by enteropathogenic Escherichia coli. J Bacteriol. 2006;188(11):3952-3961. Mutagenesis analysis has identified adhesive structures such as the common type 1 pilus (T1P), antigen 43, BFP and the EspA filament (see below) as participants in bacterial aggregation during biofilm formation on abiotic surfaces.2626 Moreira CG, Palmer K, Whiteley M, et al. Bundle-forming pili and EspA are involved in biofilm formation by enteropathogenic Escherichia coli. J Bacteriol. 2006;188(11):3952-3961.

A hallmark phenotype of both tEPEC and aEPEC is the ability to produce AE lesions.3333 Moon HW, Whipp SC, Argenzio RA, Levine MM, Giannella RA. Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines. Infect Immun. 1983;41(3):1340-1351. This phenotype is characterized by effacement of intestinal epithelial-cell microvilli and intimate adherence between the bacterium and the epithelial cell membrane. Directly beneath the adherent bacterium, marked cytoskeletal changes are seen in the epithelial cell membrane, particularly the formation of an actin-rich cup-like pedestal at the site of bacterial contact. AE lesions are observed in model EPEC infections with cultured cells and mucosal explants, as well as in intestinal biopsies from EPEC-infected infants or animals.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201.

AE lesions are encoded by LEE, which is a ∼35-kb pathogenicity island (PAI)3434 McDaniel TK, Jarvis KG, Donnenberg MS, Kaper JB. A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc Natl Acad Sci USA. 1995;92(5):1664-1668. that is organized into five operons (LEE1 to LEE5).3535 Elliott SJ, Sperandio V, Giron JA, et al. The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic Escherichia coli. Infect Immun. 2000;68(11):6115-6126.

36 Deng W, Puente JL, Grunheid S, et al. Dissecting virulence: systematic and functional analyses of a pathogenicity island. Proc Natl Acad Sci USA. 2004;101(10):3597-3602.
-3737 Dean P, Kenny B. The effector repertoire of enteropathogenic E. coli ganging up on the host cell. Curr Opin Microbiol. 2009;12(1):101-109. The LEE1, LEE2, and LEE3 operons encode components of a T3SS, and the global regulator Ler (LEE-encoded regulator).3838 Barba J, Bustamante VH, Flores-Valdez MA, Deng W, Finlay BB, Puente JL. A positive regulatory loop controls expression of the locus of enterocyte effacement-encoded regulators Ler and GrlA. J Bacteriol. 2005;187(23):7918-7930. LEE4 encodes the T3SS-secreted proteins EspA, EspB, and EspD (EPEC-secreted protein), which are components of the translocation apparatus by which other effector proteins are translocated into the cell. LEE5 encodes the adhesin intimin and its translocated receptor, Tir.3939 Kenny B, DeVinney R, Stein M, Reinscheid DJ, Frey EA, Finlay BB. Enteropathogenic Escherichia coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell. 1997;91(4):511-520.

Intimin is a 94-kDa protein encoded by the eae gene and required for intimate adherence of EPEC to host cells at the sites of AE lesions.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201. The N-terminus of intimin is highly conserved, whereas the C-terminus is highly variable.4040 Frankel G, Candy DCA, Everest P, Dougan G. Characterization of the C-terminal domains of intimin-like proteins of enteropathogenic and enterohemorrhagic Escherichia coli, Citrobacter freundii, and Hafnia alvei. Infect Immun. 1994;62(5):1835-1842. Differences in the C-terminus of intimin have been used as a basis for classification into several distinct subtypes, represented by the Greek letters α (alpha) through ζ (zeta)4141 Lacher DW, Steinsland H, Whittam TS. Allellic subtyping of the intimin locus (eae) of pathogenic Escherichia coli by fluorescent RFLP. FEMS Microbiol Lett. 2006;261:80-87.,4242 Lacher DW, Steinsland H, Blank TE, Donnenberg MS, Whittam TS. Molecular evolution of typical enteropathogenic Escherichia coli: clonal analysis by multilocus sequence typing and virulence gene allelic profiling. J Bacteriol. 2007;189(2):342-350.; the α subtype is expressed by EPEC1 strains while subtype β is associated with human EPEC2 strains. The N-terminus of intimin anchors the protein in the EPEC outer membrane, whereas the C-terminus extends from the EPEC surface and binds to the Tir. Intimin-Tir interaction leads to intimate adherence and pedestal formation beneath adherent bacteria,3939 Kenny B, DeVinney R, Stein M, Reinscheid DJ, Frey EA, Finlay BB. Enteropathogenic Escherichia coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. Cell. 1997;91(4):511-520. and inhibits NF-κB activity through tumor necrosis factor alpha (TNF-α) receptor-associated factors.4343 Ruchaud-Sparagano M-H, Muhlen S, Dean P, Kenny B. The enteropathogenic Escherichia coli (EPEC) Tir effector inhibits NF-kB activity by targeting TNFα receptor-associated factors. PLoS Pathog. 2011;7(12):e1002414. In addition to Tir, the EPEC genome contains six other LEE-encoded effector proteins that are translocated into the cell (Map, EspF, EspG, EspZ, EspH, and EspB), which interfere with different aspects of the cell's physiology.1313 Ørskov F, Whittam TS, Cravioto A, Ørskov I. Clonal relationships among classic enteropathogenic Escherichia coli (EPEC) belong to different O groups. J Infect Dis. 1990;162(1):76-81.,3636 Deng W, Puente JL, Grunheid S, et al. Dissecting virulence: systematic and functional analyses of a pathogenicity island. Proc Natl Acad Sci USA. 2004;101(10):3597-3602.,3737 Dean P, Kenny B. The effector repertoire of enteropathogenic E. coli ganging up on the host cell. Curr Opin Microbiol. 2009;12(1):101-109.,4444 Santos A, Finlay BB. Bringing down the host: enteropathogenic and enterohaemorrhagic Escherichia coli effector-mediated subversion of host innate immune pathways. Cell Microbiol. 2015;17(3):318-332.

In addition to the LEE effectors, various non-LEE (Nle)-encoded effector genes (cif, espI/nleA, nleB, nleC, nleD, nleE, nleH)3636 Deng W, Puente JL, Grunheid S, et al. Dissecting virulence: systematic and functional analyses of a pathogenicity island. Proc Natl Acad Sci USA. 2004;101(10):3597-3602.,4444 Santos A, Finlay BB. Bringing down the host: enteropathogenic and enterohaemorrhagic Escherichia coli effector-mediated subversion of host innate immune pathways. Cell Microbiol. 2015;17(3):318-332. have been described, which are located outside the EPEC LEE region, in at least six chromosomal PAIs, or in prophage elements.4545 Wong ARC, Pearson JS, Bright MD, et al. Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements. Mol Microbiol. 2011;80(6):1420-1438.,4646 Vossenkämper A, MacDonald TT, Marchès O. Always one step ahead: how pathogenic bacteria use the type III secretion system to manipulate the intestinal mucosal immune system. J Inflamm. 2011;8:11. The Nle proteins have been shown to disrupt the cytoskeleton and tight junctions of the host cell, and to modulate or prevent the host inflammatory response.4545 Wong ARC, Pearson JS, Bright MD, et al. Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements. Mol Microbiol. 2011;80(6):1420-1438.

46 Vossenkämper A, MacDonald TT, Marchès O. Always one step ahead: how pathogenic bacteria use the type III secretion system to manipulate the intestinal mucosal immune system. J Inflamm. 2011;8:11.
-4747 Raymond B, Young JC, Pallett M, Endres RG, Clements A, Frankel G. Subversion of trafficking, apoptosis, and innate immunity by type III secretion system effectors. Trends Microbiol. 2013;21(8):430-441. Although they are not required for AE lesion formation, it is understood that they contribute to increased bacterial virulence.4444 Santos A, Finlay BB. Bringing down the host: enteropathogenic and enterohaemorrhagic Escherichia coli effector-mediated subversion of host innate immune pathways. Cell Microbiol. 2015;17(3):318-332.

Intracellular tEPEC have been observed both in tissue culture and in small intestinal biopsies from an EPEC-infected infant.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201. Two studies have reported that O111:NM strains contain plasmid sequences that confer invasiveness upon E. coli K12 strains containing the cloned fragments.4848 Fletcher JN, Embaye HE, Getty B, Batt RM, Hart CA, Saunders JR. Novel invasion determinant of enteropathogenic Escherichia coli plasmid pLV501 encodes the ability to invade intestinal epithelial cells and HEp-2 cells. Infect Immun. 1992;60(6):2229-2236.,4949 Scaletsky ICA, Gatti MSV, Da Silveira FJ, DeLuca IM, Freymuller E, Travassos LR. Plasmid encoding for drug resistance and invasion of epithelial cells in enteropathogenic Escherichia coli O111:H-. Microb Pathog. 1995;18(6):387-399. Sequences homologous to these cloned genes are present in only a minority of tEPEC strains (Scaletsky et al., unpublished data).

Typical EPEC strains encode a large surface protein, lymphocyte inhibitory factor (LifA), which inhibits the expression of multiple lymphokines and inhibits lymphocyte proliferation.5050 Klapproth JM, Scaletsky ICA, McNamara BP, et al. A large toxin from pathogenic Escherichia coli strains that inhibits lymphocyte activation. Infect Immun. 2000;68(4):2148-2155. Two related genes efa1 and toxB have been implicated in adhesion to epithelial cells.5151 Nicholls L, Grant TH, Robins-Browne RM. Identification of a novel genetic locus that is required for in vitro adhesion of a clinical isolate of enterohaemorrhagic Escherichia coli to epithelial cells. Mol Microbiol. 2000;35(2):275-288.,5252 Tatsuno I, Horie M, Abe H, et al. toxB gene on pO157 of enterohemorrhagic Escherichia coli O157:H7 is required for full epithelial cell adherence phenotype. Infect Immun. 2001;69(11):6660-6669. There is evidence indicating that Efa1/LifA contributes to epithelial cell adherence in vitro5353 Badea L, Doughty S, Nicholls L, Sloan J, Robins-Browne RM, Hartland EL. Contribution of Efa/LifA to the adherence of enteropathogenic Escherichia coli to epithelial cells. Microb Pathog. 2003;34(5):205-215. and is required for intestinal colonization of mice by the related AE pathogen Citrobacter rodentium.5454 Klapproth JM, Sasaki M, Sherman M, et al. Citrobacter rodentium lifA/efa1 is essential for colonic colonization and crypt cell hyperplasia in vivo. Infect Immun. 2005;73(3):1441-1451.

Some tEPEC strains possess other fimbriae or pili in addition to BFP. Type 1 fimbriae of EPEC have been found to be antigenic in volunteer studies; however, they do not have a role in adherence to epithelial cells in vitro.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201. In addition, some EPEC strains have conserved fimbrial genes encoding homologs of long polar fimbriae (LPF),5555 Tatsuno I, Mundy R, Frankel G, et al. The lpf gene cluster for long polar fimbriae is not involved in adherence of enteropathogenic Escherichia coli or virulence of Citrobacter rodentium. Infect Immun. 2006;74(1):265-272. but a number of polymorphisms within the lpfA genes have been identified.5656 Torres AG, Kanack KJ, Tutt CB, Popov V, Kaper JB. Characterization of the second long polar (LP) fimbriae of Escherichia coli O157:H7 and distribution of LP fimbriae in other pathogenic E. coli strains. FEMS Microbiol Lett. 2004;238:333-344. Initial studies have indicated that LPF is apparently not necessary for adherence and AE lesion in human biopsies.5555 Tatsuno I, Mundy R, Frankel G, et al. The lpf gene cluster for long polar fimbriae is not involved in adherence of enteropathogenic Escherichia coli or virulence of Citrobacter rodentium. Infect Immun. 2006;74(1):265-272. The E. coli common pilus (ECP) has also been shown to act as an accessory adherence factor in EPEC, playing a role during cell adherence and/or in bacterium-bacterium interactions.5757 Saldaña Z, Erdem AL, Schuller S, et al. The Escherichia coli common pilus and the bundle-forming pilus act in concern during the formation of localized adherence by enteropathogenic E. coli. J Bacteriol. 2009;191(11):3451-3461. However, the significance of ECP to EPEC pathogenesis has not been determined. Interestingly, it has been shown that some tEPEC strains may produce a hybrid adherence phenotype in HeLa cells, i.e., LA and aggregative (AA)-like pattern concurrently (LA+/AA-like+).5858 Girão DM, Girão VBC, Irino K, Gomes TAT. Classifying Escherichia coli. Emerg Infect Dis. 2006;12(8):1297-1298. Recently, it was shown that at least some of these LA/AA-like+ strains bear large plasmids, distinct from the pEAF, that encode a so far unknown adhesin.5959 Garcia BG, Ooka T, Gotoh Y, et al. Genetic relatedness and virulence properties of enteropathogenic Escherichia coli strains of serotype O119:H6 expressing localized adherence or localized and aggregative adherence-like patterns on HeLa cells. Int J Med Microbiol. 2016;306(3):152-164. It has been proposed that the ability of such strains in producing AE lesions and an AA-associated biofilm concomitantly could worsen the patient's clinical condition, leading to persistent diarrhea.5959 Garcia BG, Ooka T, Gotoh Y, et al. Genetic relatedness and virulence properties of enteropathogenic Escherichia coli strains of serotype O119:H6 expressing localized adherence or localized and aggregative adherence-like patterns on HeLa cells. Int J Med Microbiol. 2016;306(3):152-164.

Flagella may also be involved in tEPEC adherence to epithelial cells,6060 Girón JA, Torres AG, Freer E, Kaper JB. The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol Microbiol. 2002;44(2):361-479. since certain EPEC mutants are markedly impaired in their ability to adhere and to form microcolonies. Furthermore, in one study, purified EPEC flagella and anti-flagellum antibodies were both effective in blocking the adherence of several EPEC serotypes.6060 Girón JA, Torres AG, Freer E, Kaper JB. The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol Microbiol. 2002;44(2):361-479. However, another study could not confirm the role of flagella in EPEC adherence.6161 Cleary J, Lai L-C, Shaw RK, et al. Enteropathogenic Escherichia coli (EPEC) adhesion to intestinal epithelial cells: role of bundle-forming pili (BFP), EspA filaments and intimin. Microbiology. 2004;150(3):527-538.

Some tEPEC strains harbor the astA gene, which encodes the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1).6262 Yamamoto T, Wakisaka N, Sato F, Kato A. Comparison of the nucleotide sequence of enteroaggregative Escherichia coli heat-stable enterotoxin 1 genes among diarrhea-associated Escherichia coli. FEMS Microbiol Lett. 1997;147:89-96.,6363 Dulguer MV, Fabricotti SH, Bando SY, Moreira-Filho CA, Fagundes-Neto U, Scaletsky ICA. Atypical enteropathogenic Escherichia coli strains: phenotypic and genetic profiling reveals a strong association between enteroaggregative E. coli heat-stable enterotoxin and diarrhea. J Infect Dis. 2003;188(11):1685-1694. A recent study reported that 11 of 70 (16%) tEPEC strains tested harbored an intact astA gene.6464 Silva LEP, Souza TB, Silva NP, Scaletsky ICA. Detection and genetic analysis of the enteroaggregative Escherichia coli heat-stable enterotoxin (EAST1) gene in clinical isolates of enteropathogenic Escherichia coli (EPEC) strains. BMC Microbiol. 2014;14:135. Typical EPEC strains of serotype O86:H34 produce cytolethal-distending toxin (CDT).6565 Ghilardi ACR, Gomes TAT, Trabulsi LR. Production of Cytolethal Distending Toxin and other virulence characteristics of Escherichia coli strains of serogroup O86. Mem Inst Oswaldo Cruz. 2001;96(5):703-708. The significance of EAST1 and CDT toxins in EPEC pathogenesis remains unknown.

Autotransporter (AT) proteins, which have been associated with bacterial adherence, aggregation, biofilm formation, invasion, and toxicity6666 Henderson IR, Navarro-Garcia F, Desvaux M, Fernandez RC, Ala’Aldeen D. Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev. 2004;68(4):692-744. in Gram-negative bacteria, have also been described among EPEC strains.6767 Gomes TA, Yamamoto D, Vieira MAM, Hernandes RT. Atypical enteropathogenic Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:77-96. One such protein, EspC, which is secreted by the type V secretion system and is injected by the T3SS in epithelial cells, has an IgA protease-like activity and, once in the host cytoplasm, has various cytopathic effects, including cytoskeletal damage,6868 Vidal JE, Navarro-Garcia F. EspC translocation into epithelial cells by enteropathogenic Escherichia coli requires a concerted participation of type V and III systems. Cell Microbiol. 2008;10(10):1976-1986. enhanced lysozyme resistance,7070 Salinger N, Kokona B, Fairman R, Okeke I. The plasmid-encoded regulator activates factors conferring lysozyme resistance on enteropathogenic Escherichia coli strains. Appl Environ Microbiol. 2009;75(1):275-280. hemoglobin degradation,6969 Drago-Serrano ME, Parra SG, Manjarrez-Hernández HA. EspC, an autotransporter protein secreted by enteropathogenic Escherichia coli (EPEC), displays protease activity on human hemoglobin. FEMS Microbiol Lett. 2006;265(1):35-40. hydrolysis of pepsin, factor V, and spectrin,7070 Salinger N, Kokona B, Fairman R, Okeke I. The plasmid-encoded regulator activates factors conferring lysozyme resistance on enteropathogenic Escherichia coli strains. Appl Environ Microbiol. 2009;75(1):275-280. and fodrin and focal adhesion protein degradation.7171 Navarro-Garcia F, Serapio-Palacios A, Vidal JE, Salazar MI, Tapia-Pastrana G. EspC promotes epithelial cell detachment by enteropathogenic Escherichia coli via sequential cleavages of a cytoskeletal protein and then focal adhesion proteins. Infect Immun. 2014;82(6):2255-2265. In addition, oligomerization of EspC gives rise to rope-like structures that serve as a substratum for adherence and biofilm formation as well as protecting bacteria from antimicrobial compounds.7272 Xicohtencatl-Cortes J, Saldaña Z, Deng W, et al. Bacterial macroscopic ropelike fibers with cytopathic and adhesive properties. J Biol Chem. 2010;285(42):32336-32342.

A three-stage model of tEPEC adhesion and pathogenesis, consisting of LA, signal transduction, and intimate attachment with pedestal formation, was proposed.7373 Donnenberg MS, Kaper JB. Enteropathogenic Escherichia coli. Infect Immun. 1992;60(10):3953-3961. Simultaneously with intimate attachment, a series of bacterial effector proteins are injected into host cells, where they subvert actin polymerization and other host cell processes.3737 Dean P, Kenny B. The effector repertoire of enteropathogenic E. coli ganging up on the host cell. Curr Opin Microbiol. 2009;12(1):101-109.,4444 Santos A, Finlay BB. Bringing down the host: enteropathogenic and enterohaemorrhagic Escherichia coli effector-mediated subversion of host innate immune pathways. Cell Microbiol. 2015;17(3):318-332. In the earliest stage and under correct environmental conditions, tEPEC express BFP, intimin, and the T3SS/translocon apparatus. Next, EPEC adhere to the surface of the intestinal epithelium via BFP and EspA filaments, and the T3SS injects the bacterial translocated intimin receptor (Tir) and effector proteins (EspB, EspD, EspF, EspG, and Map) directly into the host cell.3737 Dean P, Kenny B. The effector repertoire of enteropathogenic E. coli ganging up on the host cell. Curr Opin Microbiol. 2009;12(1):101-109. The effectors activate cell-signaling pathways, causing alterations in the host cell cytoskeleton and resulting in the depolymerization of actin and the loss of microvilli. Finally, bacteria intimately adhere to host cell by intimin-Tir interactions, causing a cytoskeletal rearrangement that results in pedestal-like structures. Tir promotes cytoskeletal reorganization through interaction with neural WASP (Wiskott-Aldrich syndrome protein) (N-WASP) and subsequent activation of the Arp2/3 complex,4545 Wong ARC, Pearson JS, Bright MD, et al. Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements. Mol Microbiol. 2011;80(6):1420-1438. leading to the effacement of the microvilli and the production of pedestals.4444 Santos A, Finlay BB. Bringing down the host: enteropathogenic and enterohaemorrhagic Escherichia coli effector-mediated subversion of host innate immune pathways. Cell Microbiol. 2015;17(3):318-332.,7474 Campellone KG, Giese A, Tipper DJ, Leong JM. A tyrosine-phosphorylated 12-aminoacid sequence of enteropathogenic Escherichia coli Tir binds the host adaptor protein Nck and is required for Nck localization to actin pedestals. Mol Microbiol. 2002;43(5):1227-1241. The translocated effectors disrupt host cell processes, resulting in loss of tight-junction integrity and mitochondrial function, leading to both electrolyte loss and eventual cell death.4545 Wong ARC, Pearson JS, Bright MD, et al. Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements. Mol Microbiol. 2011;80(6):1420-1438.

For actin dynamics subversion, tEPEC usually recruits Nck to the adhesion site in a Tir phosphorylated Y474-dependent mechanism. In turn, TirEHEC (enterohemorrhagic E. coli [EHEC] O157:H7) is devoid of an Y474 equivalent and employs EspFU/TccP (Tir-cytoskeleton coupling protein), a T3SS-translocated effector protein that binds N-WASP, leading to Nck-independent actin polymerization.4545 Wong ARC, Pearson JS, Bright MD, et al. Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements. Mol Microbiol. 2011;80(6):1420-1438.

aEPEC are devoid of pEAF and do not produce BFP. It is important to point out that EPEC strains of serotypes O128:H2 and O119:H2 contain a pEAF with defective bfp operons, which contain part of the bfpA gene but have the rest of the bfp gene cluster deleted. Thus, they are classified as aEPEC.1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513.,7575 Bortoloni M, Trabulsi L, Keller R, Frankel G, Sperandio V. Lack of expression of bundle-forming pili in some clinical isolates of enteropathogenic Escherichia coli (EPEC) is due to a conserved large deletion in the bfp operon. FEMS Microbiol Lett. 1999;179(1):169-174. Most aEPEC produce adherence patterns categorized as LA-like, with loosened microcolonies compared to those of the tEPEC LA pattern.1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513.,7676 Rodrigues J, Scaletsky IC, Campos LC, Gomes TA, Whittam TS, Trabulsi LR. Clonal structure and virulence factors in strains of Escherichia coli of the classic serogroup O55. Infect Immun. 1996;64(7):2680-2686.,7777 Scaletsky ICA, Pelayo JS, Giraldi R, Rodrigues J, Pedroso MZ, Trabulsi LR. EPEC adherence to HEp-2 cells. Rev Microbiol. 1996;27(Suppl 1):58-62. In addition, some isolates express the aggregative (AA) or diffuse (DA) patterns of adherence, which are characteristics of the EAEC and DAEC pathotypes, respectively,2020 Vieira MA, Andrade JR, Trabulsi LR, et al. Phenotypic and genotypic characteristics of Escherichia coli strains of non-enteropathogenic E. coli (EPEC) serogroups that carry eae and lack the EPEC adherence factor and Shiga toxin DNA probe sequences. J Infect Dis. 2001;183(5):762-772.,7878 Abe CM, Trabulsi LR, Blanco J, et al. Virulence features of atypical enteropathogenic Escherichia coli identified by the eae+ EAF-negative stx-genetic profile. Diagn Microbiol Infect Dis. 2009;64(4):357-365. or adhere in undefined patterns or are non-adherent.2020 Vieira MA, Andrade JR, Trabulsi LR, et al. Phenotypic and genotypic characteristics of Escherichia coli strains of non-enteropathogenic E. coli (EPEC) serogroups that carry eae and lack the EPEC adherence factor and Shiga toxin DNA probe sequences. J Infect Dis. 2001;183(5):762-772.,7878 Abe CM, Trabulsi LR, Blanco J, et al. Virulence features of atypical enteropathogenic Escherichia coli identified by the eae+ EAF-negative stx-genetic profile. Diagn Microbiol Infect Dis. 2009;64(4):357-365.

79 Scaletsky ICA, Aranda KR, Souza TB, Silva NP, Morais MB. Evidence of pathogenic subgroups among atypical enteropathogenic Escherichia coli strains. J Clin Microbiol. 2009;47(11):3756-3759.
-8080 Gomes TAT, Hernandes RT, Torres AG, et al. Adhesin-encoding genes from Shiga toxin-producing Escherichia coli are more prevalent in atypical than in typical enteropathogenic E. coli. J Clin Microbiol. 2011;49(11):3334-3337. Remarkably, the epithelial cell adherence phenotype displayed by aEPEC is determined in prolonged assays (6 h) of bacteria-cell interaction.1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513.,7676 Rodrigues J, Scaletsky IC, Campos LC, Gomes TA, Whittam TS, Trabulsi LR. Clonal structure and virulence factors in strains of Escherichia coli of the classic serogroup O55. Infect Immun. 1996;64(7):2680-2686.,7777 Scaletsky ICA, Pelayo JS, Giraldi R, Rodrigues J, Pedroso MZ, Trabulsi LR. EPEC adherence to HEp-2 cells. Rev Microbiol. 1996;27(Suppl 1):58-62. In addition, it has been suggested that lack of the pEAF-encoded Per proteins in the regulatory cascade of the aEPEC virulence genes may promote delayed AE lesion formation, probably making it difficult for such strains to cause disease.8181 Bueris V, Huerta-Cantillo J, Navarro-Garcia F, Ruiz RM, Cianciarullo AM, Elias WP. Late establishment of the attaching and effacing lesion caused by atypical enteropathogenic Escherichia coli depends on protein expression regulated by Per. Infect Immun. 2015;83(1):379-388.

The prevalence of intimin subtypes among aEPEC strains has been reviewed.1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149.,6767 Gomes TA, Yamamoto D, Vieira MAM, Hernandes RT. Atypical enteropathogenic Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:77-96. Intimins classified as beta1, epsilon1 and theta appear as the most frequent among aEPEC.7878 Abe CM, Trabulsi LR, Blanco J, et al. Virulence features of atypical enteropathogenic Escherichia coli identified by the eae+ EAF-negative stx-genetic profile. Diagn Microbiol Infect Dis. 2009;64(4):357-365.,8282 Gomes TAT, Irino K, Girão DM, et al. Emerging enteropathogenic Escherichia coli strains? Emerg Infect Dis. 2004;10(10):1851-1855.

83 Contreras CA, Ochoa TJ, Lacher DW, et al. Allelic variability of critical virulence genes (eae, bfpA and perA) in typical and atypical enteropathogenic Escherichia coli in Peruvian children. J Med Microbiol. 2010;59(1):25-31.

84 Xu Y, Bai X, Zhao A, et al. Genetic diversity of intimin gene of atypical enteropathogenic Escherichia coli isolated from human, animals and raw meats in China. PLoS ONE. 2016;11(3):e0152571.
-8585 Vieira MA, Santos LF, Dias RCB, et al. Atypical enteropathogenic Escherichia coli as etiologic agents of sporadic and outbreak-associated diarrhea in Brazil. J Med Microbiol. 2016;65(9):998-1006. In addition, some aEPEC strains bear adhesive-encoding genes that have been originally described in other DEC pathotypes and/or in extraintestinal pathogenic E. coli.7979 Scaletsky ICA, Aranda KR, Souza TB, Silva NP, Morais MB. Evidence of pathogenic subgroups among atypical enteropathogenic Escherichia coli strains. J Clin Microbiol. 2009;47(11):3756-3759.,8080 Gomes TAT, Hernandes RT, Torres AG, et al. Adhesin-encoding genes from Shiga toxin-producing Escherichia coli are more prevalent in atypical than in typical enteropathogenic E. coli. J Clin Microbiol. 2011;49(11):3334-3337.,8282 Gomes TAT, Irino K, Girão DM, et al. Emerging enteropathogenic Escherichia coli strains? Emerg Infect Dis. 2004;10(10):1851-1855.,8686 Tennant SM, Tauschek M, Azzopardi K, et al. Characterisation of atypical enteropathogenic E. coli strains of clinical origin. BMC Microbiol. 2009;9:117.

87 Scaletsky ICA, Aranda KRS, Souza TB, Silva NP. Adherence factors in atypical enteropathogenic Escherichia coli strains expressing the localized adherence-like pattern in HEp-2 cells. J Clin Microbiol. 2010;48(1):302-306.
-8888 Hernandes RT, Velsko I, Sampaio SCF, et al. Fimbrial adhesins produced by atypical enteropathogenic Escherichia coli strains. Appl Environ Microbiol. 2011;77(23):8391-8399. This observation suggests that aEPEC could employ additional adherence mechanisms besides the Tir-intimin interaction. The only adhesin first characterized in an aEPEC strain (serotype O26:H11) is the locus of diffuse adherence (LDA), which is an afimbrial adhesin that confers the diffuse pattern of adherence on HEp-2 cells, when cloned in E. coli K-12 strains.8989 Scaletsky IC, Michalski J, Torres AG, Dulguer MV, Kaper JB. Identification and characterization of the locus for diffuse adherence, which encodes a novel afimbrial adhesin found in atypical enteropathogenic Escherichia coli. Infect Immun. 2005;73(8):4753-4765. The T3SS-translocon has been also shown to contribute to the adherence efficacy of an aEPEC strain in vitro.9090 Hernandes RT, Miguel A, Yamamato D, et al. Dissection of the role of pili and Type 2 and 3 secretion systems in adherence and biofilm formation of an atypical enteropathogenic Escherichia coli strain. Infect Immun. 2013;81(23):3793-3802. The prevalence of these different adhesins among aEPEC has been recently reviewed.1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149.,6767 Gomes TA, Yamamoto D, Vieira MAM, Hernandes RT. Atypical enteropathogenic Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:77-96.,9191 Hu J, Torres AG. Enteropathogenic Escherichia coli: foe or innocent bystander? Clin Microbiol Infect. 2015;21(8):729-734.

Moreover, it has been recently shown that the flagellar cap protein FliD of an aEPEC strain (serotype O51:H40) binds to unknown receptors on intestinal Caco-2 cell microvilli.9292 Sampaio SCF, Luiz WB, Vieira MAM, et al. Flagellar cap protein FliD mediates adherence of atypical enteropathogenic Escherichia coli to enterocyte microvilli. Infect Immun. 2016;84(4):1112-1122. Interestingly, an anti-FliD serum and purified FliD reduced adherence of the aEPEC as well as that of tEPEC, EHEC and ETEC prototype strains to the same cell line.9292 Sampaio SCF, Luiz WB, Vieira MAM, et al. Flagellar cap protein FliD mediates adherence of atypical enteropathogenic Escherichia coli to enterocyte microvilli. Infect Immun. 2016;84(4):1112-1122. Furthermore, it has been suggested that adherence of aEPEC of serotype O26:H11 may be mediated by binding of the flagellin protein FliC (the subunit of the flagella shaft) to cellular fibronectin.9393 Moraes CTP, Polatto JM, Rossato SS, et al. Flagellin and GroEL mediates in vitro binding of an atypical enteropathogenic Escherichia coli to cellular fibronectin. BMC Microbiol. 2015;15:278. However, the role of the flagella in aEPEC in vivo colonization has yet to be investigated.

Atypical EPEC strains have also been shown to adhere to abiotic surfaces (polystyrene and glass).9494 Culler HF, Mota CM, Abe CM, Elias WP, Sircili MP, Franzolin MR. Atypical enteropathogenic Escherichia coli strains form biofilm on abiotic surfaces regardless of their adherence pattern on cultured epithelial cells. Biomed Res Int. 2014;2014:845147.,9595 Nascimento HH, Silva LEP, Souza RT, Silva NP, Scaletsky ICA. Phenotypic and genotypic characteristics associated with biofilm formation in clinical isolates of atypical enteropathogenic Escherichia coli (aEPEC) strains. BMC Microbiol. 2014;14:184. The non-fimbrial adhesin curli and the T1P have been shown to mediate binding to these surfaces in some aEPEC at different temperatures.9090 Hernandes RT, Miguel A, Yamamato D, et al. Dissection of the role of pili and Type 2 and 3 secretion systems in adherence and biofilm formation of an atypical enteropathogenic Escherichia coli strain. Infect Immun. 2013;81(23):3793-3802.,9696 Weiss-Muszkat M, Shakh D, Zhou Y, et al. Biofilm formation by and multicellular behavior of Escherichia coli O55:H7, an atypical enteropathogenic strain. Appl Environ Microbiol. 2010;76(5):1545-1554.

The LEE region of some aEPEC strains display a genetic organization similar to that found in the tEPEC prototype E2348/69 strain.9797 Gärtner JF, Schmidt MA. Comparative analysis of locus of enterocyte effacement pathogenicity islands of atypical enteropathogenic Escherichia coli. Infect Immun. 2004;72(11):6722-6728. Although the T3SS-encoding genes are considerably conserved,9797 Gärtner JF, Schmidt MA. Comparative analysis of locus of enterocyte effacement pathogenicity islands of atypical enteropathogenic Escherichia coli. Infect Immun. 2004;72(11):6722-6728.,9898 Ingle DJ, Tauschek M, Edwards DJ, et al. Evolution of atypical enteropathogenic E. coli by repeated acquisition of LEE pathogenicity island variants. Nat Microbiol. 2016;1:15010. the effector protein-encoding genes display important differences, and remarkable differences can be detected at the 5′ and 3′ flanking regions of aEPEC, suggesting the occurrence of different evolutionary events.9999 Müller D, Benz I, Liebchen A, Gallitz I, Karch H, Schmidt MA. Comparative analysis of the locus of enterocyte effacement and its flanking regions. Infect Immun. 2009;77(8):3501-3513. Atypical EPEC strains may carry two tccP variants, tccP and/or tccP2, suggesting that some aEPEC strains may use both Tir-Nck and Tir-TccP pathways to promote actin polymerization.100100 Ooka T, Vieira MA, Ogura Y, et al. Characterization of tccP2 carried by atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2007;271(1):126-135. Interestingly, Rocha and colleagues101101 Rocha SPD, Abe CM, Sperandio V, Bando SY, Elias WP. Atypical enteropathogenic Escherichia coli that contains functional locus of enterocyte effacement genes can be attaching-and-effacing negative in cultured epithelial cells. Infect Immun. 2011;79(5):1833-1841. showed that transformation of a non-adherent aEPEC strain (serotype O88:HNM) with a TccP expressing-plasmid, conferred this strain the ability to adhere to and to induce actin-accumulation in HeLa cells.

The occurrence and prevalence of Nle in aEPEC strains have been recently reviewed.6767 Gomes TA, Yamamoto D, Vieira MAM, Hernandes RT. Atypical enteropathogenic Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:77-96. It has been suggested that different isolates can employ distinct strategies to promote damage to the host and cause disease.4545 Wong ARC, Pearson JS, Bright MD, et al. Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements. Mol Microbiol. 2011;80(6):1420-1438. In addition, the Nle effectors Ibe (invasion of endothelial cells) and EspT have been originally described and characterized in aEPEC strains.102102 Bulgin R, Arbeloa A, Goulding D, et al. The T3SS effector EspT defines a new category of invasive enteropathogenic E. coli (EPEC) which form intracellular actin pedestals. PLoS Pathog. 2009;5:e1000683.,103103 Buss C, Müller D, Rüter C, Heusipp G, Schmidt MA. Identification and characterization of Ibe, a novel type III effector protein of A/E pathogens targeting human IQGAP1. Cell Microbiol. 2009;11(4):661-677. Ibe appears to regulate Tir phosphorylation and to enhance actin polymerization and pedestal formation,103103 Buss C, Müller D, Rüter C, Heusipp G, Schmidt MA. Identification and characterization of Ibe, a novel type III effector protein of A/E pathogens targeting human IQGAP1. Cell Microbiol. 2009;11(4):661-677. while EspT104104 Arbeloa A, Blanco M, Moreira FC, et al. Distribution of espM and espT among enteropathogenic and enterohaemorrhagic Escherichia coli. J Med Microbiol. 2009;58(8):988-995. modulates actin dynamics, leading to membrane ruffling and cell invasion, and induces macrophages to produce interleukins IL-8 and IL-1β and PGE2.102102 Bulgin R, Arbeloa A, Goulding D, et al. The T3SS effector EspT defines a new category of invasive enteropathogenic E. coli (EPEC) which form intracellular actin pedestals. PLoS Pathog. 2009;5:e1000683.

Invasion of epithelial cells in vitro in an intimin-dependent pathway has been described in an aEPEC strain,105105 Hernandes RT, Silva RM, Carneiro SM, et al. The localized adherence pattern of an atypical enteropathogenic Escherichia coli is mediated by intimin omicron and unexpectedly promotes HeLa cell invasion. Cell Microbiol. 2008;10(2):415-425. but further studies pointed out that the invasive phenotype is not a common characteristic among aEPEC.106106 Pacheco VCR, Yamamoto D, Abe CM, et al. Invasion of differentiated intestinal Caco-2 cells is a sporadic property among atypical enteropathogenic Escherichia coli strains carrying common intimin subtypes. Pathog Dis. 2014;70(2):167-175. Despite their invasive potential in vitro,107107 Yamamoto D, Hernandes RT, Blanco M, et al. Invasiveness as a putative additional virulence mechanism of some atypical enteropathogenic Escherichia coli strains with different uncommon intimin types. BMC Microbiol. 2009;9:146. most aEPEC are considered extracellular pathogens.55 Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26(4):822-880.

It has been shown that apical infection of cultured human mucin-secreting intestinal HT29-MTX cells by some aEPEC strains may induce increased production of secreted MUC2 and MUC5AC mucins and membrane-bound MUC3 and MUC4 mucins.108108 Vieira MAM, Salvador FA, Silva RM, et al. Prevalence and characteristics of the O122 pathogenicity island in typical and atypical enteropathogenic Escherichia coli strains. J Clin Microbiol. 2010;48(4):1452-1455. This observation suggests that the apically adhering bacteria could exploit large amounts of mucins to grow more efficiently in the host intestines, characterizing a putative new virulence mechanism in aEPEC.108108 Vieira MAM, Salvador FA, Silva RM, et al. Prevalence and characteristics of the O122 pathogenicity island in typical and atypical enteropathogenic Escherichia coli strains. J Clin Microbiol. 2010;48(4):1452-1455.

AT proteins have also been shown to be produced by some aEPEC strains.6767 Gomes TA, Yamamoto D, Vieira MAM, Hernandes RT. Atypical enteropathogenic Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:77-96. Abreu and collegues109109 Abreu AG, Bueris V, Porangaba TM, Sircili MP, Navarro-Garcia F, Elias WP. Autotransporter protein-encoding genes of diarrheagenic Escherichia coli are found in both typical and atypical enteropathogenic E. coli strains. Appl Environ Microbiol. 2013;79(21):411-414. have shown that the AT protein encoded by the ehaC gene, which is involved in biofilm formation in EHEC strains, was the most frequent, with a significantly higher prevalence than in tEPEC. Although the prevalence of the AT protein Pic (protein involved in intestinal colonization), formerly identified in EAEC, is not a common finding in aEPEC strains, it also appears to mediate colonization of mouse intestines, hemagglutination, mucin cleavage, and complement components degradation.110110 Abreu AG, Abe CM, Nunes KO, et al. The serine protease Pic as a virulence factor of atypical enteropathogenic Escherichia coli. Gut Microbes. 2016;7(2):115-125. More recently, some aEPEC strains were shown to cause cell damage by secreting the AT protein Pet (plasmid encoded toxin) to the extracellular environment.111111 Ruiz RC, Melo KCM, Rossato SS, et al. Atypical enteropathogenic Escherichia coli secretes plasmid encoded toxin. Biomed Res Int. 2014;2014:896235.

Epidemiology

The prevalence of EPEC infections varies between epidemiological studies on the basis of differences in study populations, age distributions, and methods (serotyping, adherence patterns, and presence of the eae or conserved LEE genes) used for detection and diagnosis.112112 Ochoa TJ, Barletta F, Contreras C, Mercado E. New insights into the epidemiology of enteropathogenic Escherichia coli infection. Trans R Soc Trop Med Hyg. 2008;102(9):852-856. In addition, differences in geographic regions, periods of time and socioeconomic class may also contribute to differences in the epidemiology of EPEC-induced diarrheal disease.113113 Maranhão HS, Medeiros MCC, Scaletsky ICA, Fagundes-Neto U, Morais MB. The epidemiological and clinical characteristics and nutritional development of infants with acute diarrhea, in northeastern Brazil. Ann Trop Med Parasitol. 2008;102(4):357-365. Lack of discrimination between tEPEC and aEPEC in some studies also makes such analysis difficult.

Diarrhea due to tEPEC decreases with age, and infections in adults are rarely reported. This apparent resistance in adults and older children has been attributed to the loss of specific receptors with age or development of immunity.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201.

For many decades, studies conducted worldwide have shown that tEPEC serotypes are strongly associated with diarrhea in children <1 year of age, mainly in poor children in urban centers.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201.,1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513.,1515 Gomes TAT, González-Pedrajo B. Enteropathogenic Escherichia coli (EPEC). In: Torres AG, ed. Pathogenic Escherichia coli in Latin America. Sharjah, United Arab Emirates: Betham Science Publishers Ltd.; 2010:66-126. The association with diarrhea was particularly strong in infants less than 6 months of age. Studies in Brazil, Chile, Mexico, and South Africa, showed that 30-40% of infantile diarrhea cases were due to tEPEC serotypes.1515 Gomes TAT, González-Pedrajo B. Enteropathogenic Escherichia coli (EPEC). In: Torres AG, ed. Pathogenic Escherichia coli in Latin America. Sharjah, United Arab Emirates: Betham Science Publishers Ltd.; 2010:66-126.,112112 Ochoa TJ, Barletta F, Contreras C, Mercado E. New insights into the epidemiology of enteropathogenic Escherichia coli infection. Trans R Soc Trop Med Hyg. 2008;102(9):852-856.,114114 Gomes TAT, Vieira MAM, Wachsmuth IK, Blake PA, Trabulsi LR. Serotype-specific prevalence of Escherichia coli strains with EPEC adherence factor genes in infants with and without diarrhea in São Paulo, Brazil. J Infect Dis. 1989;160(1):131-135. However, the epidemiology of EPEC infections has shifted. In numerous developing countries, where the prevalence of EPEC infection had been high until the 1990s, recent studies have not identified a significant association between tEPEC and infantile diarrhea. In Brazil, 92% of EPEC isolates collected from children between 2001 and 2002 were atypical,115115 Franzolin MR, Alves RCB, Keller R, et al. Prevalence of diarrheagenic Escherichia coli in children with diarrhea in Salvador, Bahia, Brazil. Mem Inst Oswaldo Cruz. 2005;100(4):359-363. compared to 38% in a 1998-1999 study.7979 Scaletsky ICA, Aranda KR, Souza TB, Silva NP, Morais MB. Evidence of pathogenic subgroups among atypical enteropathogenic Escherichia coli strains. J Clin Microbiol. 2009;47(11):3756-3759. However, other studies still report tEPEC being more prevalent than aEPEC as a cause of diarrhea.116116 Alikhani MY, Mirsalehian A, Aslani MM. Detection of typical and atypical enteropathogenic Escherichia coli (EPEC) in Iranian children with and without diarrhea. J Med Microbiol. 2006;55(9):1159-1163. In addition, in some less developed areas (Africa and Asia), tEPEC are still some of the most important enteropathogens.117117 Rajendran P, Ajjampur SSR, Chidambaram D, et al. Pathotypes of diarrheagenic Escherichia coli in children attending a tertiary care hospital in South India. Diagn Microbiol Infect Dis. 2010;68(2):117-122.

118 Kotloff KL, Nataro JP, Blackwelder W, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicentre Study, GEMS): a prospective, case-control study. Lancet. 2013;382(9888):209-222.

119 Santona S, Diaz N, Fiori PL, et al. Genotypic and phenotypic features of enteropathogenic Escherichia coli isolated in industrialized and developing countries. J Infect Dev Ctries. 2013;7(3):214-219.

120 Nejma BSB, Hassine Zaafrane M, Hassine F, et al. Etiology of acute diarrhea in tunisian children with emphasis on diarrheagenic Escherichia coli: prevalence and identification of E. coli virulence markers. Iran J Public Health. 2014;43(7):947-960.

121 Langendorf C, Le Hello S, Moumouni A, et al. Enteric bacterial pathogens in children with diarrhea in Niger: diversity and antimicrobial resistance. PLoS ONE. 2015;10(3):e0120275.
-122122 Odetoyin B, Hofmann J, Aboderin A, Okeke I. Diarrhoeagenic Escherichia coli in mother-child pairs in Ile-Ife, South Western Nigeria. BMC Infect Dis. 2016;28. Based on the recently completed Global Enteric Multicenter Study (GEMS) involving children less than 5 years of age from seven sites in Africa and Asia, tEPEC was significantly associated with moderate to severe diarrhea in children under 2 years of age in Kenya, whereas aEPEC was not associated with this type of diarrhea.118118 Kotloff KL, Nataro JP, Blackwelder W, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicentre Study, GEMS): a prospective, case-control study. Lancet. 2013;382(9888):209-222.

Transmission of tEPEC follows a fecal-oral process through contaminated surfaces, weaning fluids, and human carriers.123123 Levine MM, Edelman R. Enteropathogenic Escherichia coli of classic serotypes associated with infant diarrhea: epidemiology and pathogenesis. Epidemiol Rev. 1984;6:31-51. Although rare, outbreaks among adults seem to occur through the ingestion of contaminated food and water; however, no specific environmental reservoir has been identified.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201. The infective dose in adult volunteers is high, at 108 to 1010 organisms,124124 Levine MM, Bergquist EJ, Nalin DR, et al. Escherichia coli strains that cause diarrhea but do not produce heat-labile or heat-stable enterotoxins and are non-invasive. Lancet. 1978;1(8074):1119-1122. while the infective dose that causes disease in children is unknown. EPEC outbreaks have been reported to show a seasonal distribution with peaks during the warm months.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201.,125125 Behiry IK, Abada EA, Ahmed EA, Labeed RS. Enteropathogenic Escherichia coli associated with diarrhea in children in Cairo, Egypt. Sci World J. 2011;11:2613-2619. Humans are the only known reservoir for tEPEC, with symptomatic and asymptomatic children and asymptomatic adults being the most likely source.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201.

In contrast to tEPEC, aEPEC have been found in diarrheic patients of all ages and in adults with HIV-AIDS.8282 Gomes TAT, Irino K, Girão DM, et al. Emerging enteropathogenic Escherichia coli strains? Emerg Infect Dis. 2004;10(10):1851-1855.,126126 Lozer DM, Souza TB, Monfardini MV, et al. Genotypic and phenotypic analysis of diarrheagenic Escherichia coli strains isolated from Brazilian children living in low socioeconomic level communities. BMC Infect Dis. 2013;13:418.

127 Assis FEA, Wolf S, Surek M, et al. Impact of Aeromonas and diarrheagenic Escherichia coli screening in patients with diarrhea in Paraná, Southern Brazil. J Infect Dev Ctries. 2014;8(12):1609-1614.
-128128 Dias RCB, Santos BC, Santos LF, et al. Diarrheagenic Escherichia coli pathotypes investigation revealed atypical enteropathogenic E. coli as putative emerging diarrheal agents in children living in Botucatu, São Paulo State, Brazil. APMIS. 2016;124:299-308. Furthermore, the proportion of aEPEC strains has increased, and aEPEC strains have outnumbered tEPEC strains and have also been associated with childhood diarrhea in some developing and developed countries.1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513.,1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149.,6767 Gomes TA, Yamamoto D, Vieira MAM, Hernandes RT. Atypical enteropathogenic Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:77-96.,9191 Hu J, Torres AG. Enteropathogenic Escherichia coli: foe or innocent bystander? Clin Microbiol Infect. 2015;21(8):729-734.,112112 Ochoa TJ, Barletta F, Contreras C, Mercado E. New insights into the epidemiology of enteropathogenic Escherichia coli infection. Trans R Soc Trop Med Hyg. 2008;102(9):852-856.,129129 Foster MA, Iqbal J, Zhang C, et al. Enteropathogenic and enteroaggregative E. coli in stools of children with acute gastroenteritis in Davidson County, Tennessee. Diagn Microbiol Infect Dis. 2015;83(3):319-324. However, the increase in prevalence of aEPEC may also reflect the refined discrimination between tEPEC and aEPEC.1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513.,1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149.,9191 Hu J, Torres AG. Enteropathogenic Escherichia coli: foe or innocent bystander? Clin Microbiol Infect. 2015;21(8):729-734.

The role of aEPEC in diarrhea is not clear because of its detection at similar rates in both diarrheic and non-diarrheic patients in various geographical areas.1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149.,9191 Hu J, Torres AG. Enteropathogenic Escherichia coli: foe or innocent bystander? Clin Microbiol Infect. 2015;21(8):729-734.,128128 Dias RCB, Santos BC, Santos LF, et al. Diarrheagenic Escherichia coli pathotypes investigation revealed atypical enteropathogenic E. coli as putative emerging diarrheal agents in children living in Botucatu, São Paulo State, Brazil. APMIS. 2016;124:299-308. In studies conducted in the last five years, aEPEC have been found at rates varying from ∼0.05 to ∼12% in diarrheic versus 0 to ∼14% in non-diarrheic patients.6767 Gomes TA, Yamamoto D, Vieira MAM, Hernandes RT. Atypical enteropathogenic Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:77-96. Some recent studies have also implicated aEPEC as the cause of persistent and bloody diarrhea.1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149.,9191 Hu J, Torres AG. Enteropathogenic Escherichia coli: foe or innocent bystander? Clin Microbiol Infect. 2015;21(8):729-734. Moreover, aEPEC strains have been associated with diarrheal outbreaks in Finland, United States, Japan, China1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149.,9191 Hu J, Torres AG. Enteropathogenic Escherichia coli: foe or innocent bystander? Clin Microbiol Infect. 2015;21(8):729-734.,112112 Ochoa TJ, Barletta F, Contreras C, Mercado E. New insights into the epidemiology of enteropathogenic Escherichia coli infection. Trans R Soc Trop Med Hyg. 2008;102(9):852-856. and Brazil.8585 Vieira MA, Santos LF, Dias RCB, et al. Atypical enteropathogenic Escherichia coli as etiologic agents of sporadic and outbreak-associated diarrhea in Brazil. J Med Microbiol. 2016;65(9):998-1006.

In contrast to tEPEC, which are seldom found in animals,1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513. many aEPEC strains have been found in both diarrheic and healthy animals.1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149.,6767 Gomes TA, Yamamoto D, Vieira MAM, Hernandes RT. Atypical enteropathogenic Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:77-96. Interestingly, animal aEPEC serogroups associated with human diarrhea have been identified (e.g., O26, O103, O119, O128, O142 and O157).1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149.,130130 Kolenda R, Burdukiewicz M, Schierack P. A systematic review and meta-analysis of the epidemiology of pathogenic Escherichia coli of calves and the role of calves as reservoirs for human pathogenic E. coli. Front Cell Infect Microbiol. 2015;5:23.,131131 Moura RA, Sircili MP, Leomil L, et al. Clonal Relationship among Atypical enteropathogenic Escherichia coli strains isolated from different animal species and humans. Appl Environ Microbiol. 2009;75(23):7399-7408. Serotyping and molecular methods such as multilocus sequence typing (MSLT) and pulsed field gel electrophoresis (PFGE) have contributed to demonstrating that domestic and wild animals and the environment are potential sources of aEPEC for human infections in several regions.1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149.,6767 Gomes TA, Yamamoto D, Vieira MAM, Hernandes RT. Atypical enteropathogenic Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:77-96.,9191 Hu J, Torres AG. Enteropathogenic Escherichia coli: foe or innocent bystander? Clin Microbiol Infect. 2015;21(8):729-734.,131131 Moura RA, Sircili MP, Leomil L, et al. Clonal Relationship among Atypical enteropathogenic Escherichia coli strains isolated from different animal species and humans. Appl Environ Microbiol. 2009;75(23):7399-7408. Therefore, although no direct transmission from animals to humans has been shown so far, it is reasonable to suggest that some aEPEC strains are potentially zoonotic pathogens, with a large variety of animal species serving as important reservoirs.6767 Gomes TA, Yamamoto D, Vieira MAM, Hernandes RT. Atypical enteropathogenic Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:77-96.,9191 Hu J, Torres AG. Enteropathogenic Escherichia coli: foe or innocent bystander? Clin Microbiol Infect. 2015;21(8):729-734. In addition, foods including raw meat, pasteurized milk and vegetables and water have also been implicated as vehicles of aEPEC in human infections.6767 Gomes TA, Yamamoto D, Vieira MAM, Hernandes RT. Atypical enteropathogenic Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:77-96.

aEPEC strains comprise a very assorted group with various additional virulence mechanisms that altogether can modulate the disease outcome or their occurrence in asymptomatic persons. There have been continuous advances in our knowledge of the genetic background and pathogenicity of aEPEC as well as in the information gathered from epidemiological studies, and may contribute to the discrimination between strains that cause diarrhea and those that cause asymptomatic infections.

Detection and diagnosis

EPEC can be detected by DNA probes or PCR assays using primers targeting the eae and stx genes.132132 Gannon VP, Rashed M, King RK, Thomas EJ. Detection and characterization of the eae gene of Shiga-like toxin-producing Escherichia coli using polymerase chain reaction. J Clin Microbiol. 1993;31(5):1268-1274.,133133 Karch H, Böhm H, Schmidt H, Gunzer F, Aleksic S, Heesemann J. Clonal structure and pathogenicity of Shiga-like toxin-producing, sorbitol-fermenting Escherichia coli O157:H-. J Clin Microbiol. 1993;31(5):1200-1205. All eae-positive and stx-negative E. coli strains are further tested by PCR for the presence of the bfpA gene encoding bundlin66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201. and/or the EAF plasmid to differentiate tEPEC from aEPEC.134134 Gunzburg TS, Tornieporth GN, Riley WL. Identification of enteropathogenic Escherichia coli by PCR-based detection of the bundle-forming pilus gene. J Clin Microbiol. 1995;33(5):1375-1377.,135135 Franke J, Franke S, Schmidt H, et al. Nucleotide sequence analysis of enteropathogenic Escherichia coli (EPEC) adherence factor probe and development of PCR for rapid detection of EPEC harboring virulence plasmids. J Clin Microbiol. 1994;32(10):2460-2463. However, this may fail to identify all bfpA-positive EPEC strains, since multiple alleles of bfpA have been identified,136136 Blank TE, Zhong H, Bell AL, Whittam TS, Donnenberg MS. Molecular variation among type IV pilin (bfpA) genes from diverse enteropathogenic Escherichia coli strains. Infect Immun. 2000;68(12):7028-7038. suggesting that some current PCR methods may fail to identify all bfpA-positive EPEC strains.

However, in routine microbiology laboratories, all E. coli colonies obtained from primary isolation plates are traditionally screened by slide agglutination assays using sera against the classical EPEC serogroups O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O158.137137 World Health Organization. Programme for control of diarrhoeal diseases (CDD/83.3 Rev.1). In: Manual for Laboratory Investigation of Acute Enteric Infections. Geneva, Sweden: World Health Organization; 1987:27. This method is practical and easy to perform, the main advantage of which is the commercial availability of the sera. However, the disadvantage of this method is the heterogeneity of EPEC serogroups that can comprise categories other than EPEC, the inability to distinguish tEPEC from aEPEC within these serogroups, and the occurrence of EPEC strains belonging to serogroups other than the classical EPEC serogroups.1212 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8(5):508-513.,1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149.,138138 Trabulsi LR, Campos LC, Whittam TS, Gomes TAT, Rodrigues J, Gonçalves AG. Traditional and non-traditional enteropathogenic Escherichia coli serogroups. In: São Paulo SP, ed. International Symposium on Enteropathogenic Escherichia coli (EPEC), Revista de Microbiologia (Impresso) (Cessou em 1999. Cont. ISSN 1517-8382 Brazilian Journal of Microbiology (Impresso)). vol. 27. São Paulo: Sociedade Brasileira de Microbiologia; 1996:1-6.,139139 Piazza RMF, Abe CM, Horton DSPQ, et al. Detection and subtyping methods of diarrheagenic Escherichia coli strains. In: Torres AG, ed. Pathogenic Escherichia coli in Latin America. Bentham Science Publishers; 2010:95-115.

Since EPEC strains are defined based on their virulence properties, a set of proteins, including intimin, BFP and T3SS secreted proteins can be considered targets for diagnosis. BFP expression has been considered the phenotypic marker of tEPEC.1818 Hernandes RT, Elias WP, Vieira AM, Gomes TAT. An overview of atypical enteropathogenic Escherichia coli. FEMS Microbiol Lett. 2009;297:137-149.,7878 Abe CM, Trabulsi LR, Blanco J, et al. Virulence features of atypical enteropathogenic Escherichia coli identified by the eae+ EAF-negative stx-genetic profile. Diagn Microbiol Infect Dis. 2009;64(4):357-365.,140140 Nara JM, Cianciarullo AM, Culler HF, et al. Differentiation of typical and atypical enteropathogenic Escherichia coli using colony immunoblot for detection of bundle-forming pilus expression. J Appl Microbiol. 2010;109(1):35-43. Immunofluorescence and immunoblotting tests using monoclonal or polyclonal antibodies against BFP have been employed.141141 Girón JA, Qadri F, Azim T, Jarvis KJ, Kaper JB, Albert MJ. Monoclonal antibodies specific for the bundle-forming pilus of enteropathogenic Escherichia coli. Infect Immun. 1995;63:4949-4952.,142142 Gismero-Ordoñez J, Dall’agnol M, Trabulsi LR, Girón JA. Expression of the bundle-forming pilus by enteropathogenic Escherichia coli strains of heterologous serotypes. J Clin Microbiol. 2002;40(6):2291-2296. These cited authors detected the production of BFP on different media, in which they reported that 91% of the tEPEC strains tested produced BFP in Dulbecco's Modified Eagle Medium (DMEM), 89% in MacConkey, and 83% in EMB agars. These results are particularly interesting, since MacConkey and EMB agars are routinely used for the identification of lactose-fermenting E. coli isolated from diarrheal stools. A colony immunoblot assay for tEPEC detection based on BFP expression was also standardized using a rabbit tEPEC anti-BFP polyclonal serum. Standardization was done after growing the bacterial isolates on DMEM agar containing fetal bovine serum or tryptic soy agar containing 5% washed sheep blood (TSAB). This test showed a positivity of 92 and 83% and specificity of 96 and 97%, respectively, when the culture was done in DMEM and TSAB. This method combines the simplicity of an immunoserological assay with the high efficiency of testing a large number of EPEC colonies.140140 Nara JM, Cianciarullo AM, Culler HF, et al. Differentiation of typical and atypical enteropathogenic Escherichia coli using colony immunoblot for detection of bundle-forming pilus expression. J Appl Microbiol. 2010;109(1):35-43.

Concerning intimin detection, a rabbit polyclonal sera raised against the conserved region of intimin (Int388-667)143143 Adu-Bobie J, Frankel G, Bain C, et al. Detection of intimins α, β, γ, and δ, four intimin derivatives expressed by attaching and effacing microbial pathogens. J Clin Microbiol. 1998;36(3):662-668. was employed in order to detect tEPEC isolates expressing α, β, γ, δ and ɛ intimin reported an application of immunoblotting with 100% specificity and 97% sensitivity in the detection of eae positive E. coli strains.144144 Batchelor M, Knutton S, Caprioli A, et al. Development of a universal intimin antiserum and PCR primers. J Clin Microbiol. 1999;37(12):3822-3827.

145 Koga PCM, Menezes CA, Lima FA, et al. Polyclonal anti-intimin antibody: immunological characterization and its use in EPEC diagnosis. Braz J Microbiol. 2003;34(1):5-7.
-146146 Menezes MA, Rocha LB, Koga PCM, et al. Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin. J Appl Microbiol. 2010;108(3):878-887. These authors clearly demonstrated that polyclonal rabbit antisera is suitable for immunoblotting as a diagnostic tool, and showed that protein denaturation and linearization is a critical step for anti-intimin antibody accessibility. Indeed, even employing the recombinant antibody such as single chain fragment variable (scFv-intimin),147147 Menezes MA, Aires KA, Ozaki CY, et al. Cloning approach and functional analysis of anti-intimin single-chain variable fragment (scFv). BMC Research Notes. 2011;4:30.,148148 Caravelli A, Luz DE, Andrade FB, Moraes CT, Maranhão AQ, Piazza RM. Sensitive and specific detection of enteropathogenic and enterohemorrhagic Escherichia coli using recombinant anti-intimin antibody by immunofluorescence assay. Diagn Microbiol Infect Dis. 2013;77(4):301-303. merely by immunofluorescence the scFv-intimin was able to detect tEPEC, aEPEC, and EHEC isolates, showing that intimin can be a target for EPEC and EHEC diagnosis after bacterial permeabilization.148148 Caravelli A, Luz DE, Andrade FB, Moraes CT, Maranhão AQ, Piazza RM. Sensitive and specific detection of enteropathogenic and enterohemorrhagic Escherichia coli using recombinant anti-intimin antibody by immunofluorescence assay. Diagn Microbiol Infect Dis. 2013;77(4):301-303.

Regarding secreted proteins, Lu et al.149149 Lu Y, Toma C, Honma Y, Iwanaga M. Detection of EspB using reversed passive latec agglutination: application to determination of enteropathogenic Escherichia coli. Diagn Microbiol Infect Dis. 2002;43(1):7-12. developed a new practical method to identify EPEC by detecting the E. coli secreted protein B (EspB) in the culture supernatant by reversed passive latex agglutination (RPLA), after the strains have been cultivated in DMEM. In addition, Nakasone et al.,150150 Nakasone N, Toma C, Lu Y, Iwanaga M. Development of a rapid immunochromatographic test to identify enteropathogenic and enterohemorrhagic Escherichia coli by detecting EspB. Diagn Microbiol Infect Dis. 2007;57(1):21-25. established a rapid immunochromatographic (IC) test to identify the presence of EspB in EPEC and EHEC isolates. The detection limit of the test has been reported to be 4 ng/mL, and the results showed 96.9% sensitivity and 100% specificity. The IC test for the detection of EspB may be a practical method to define EPEC or EHEC both in clinical laboratories and the field.150150 Nakasone N, Toma C, Lu Y, Iwanaga M. Development of a rapid immunochromatographic test to identify enteropathogenic and enterohemorrhagic Escherichia coli by detecting EspB. Diagn Microbiol Infect Dis. 2007;57(1):21-25.

In addition, a rapid agglutination test using latex beads coated with anti-EspB mAb was standardized, showing 97% sensitivity, 98% specificity and 97% efficiency, which is required for the diagnosis of enteropathogenic diseases and can be employed in developing countries with poorly equipped laboratories.151151 Rocha LB, Santos AR, Munhoz DD, et al. Development of a rapid agglutination latex test for diagnosis of enteropathogenic and enterohemorrhagic Escherichia coli infection in developing world: defining the biomarker, antibody and method. PLoS Negl Trop Dis. 2014;8(9):e3150.

Enterohemorrhagic (Shiga toxin-producing) E. coli (EHEC/STEC)

EHEC/STEC represent a well-known group of foodborne pathogens distributed worldwide. The ability to produce one or more of the Shiga toxin (Stx) family cytotoxins152152 Melton-Celsa AR. Shiga toxin (Stx) classification, structure, and function. Microbiol Spectr. 2014;2(3). EHEC-0024-2013. constitutes the main virulence attribute of this pathogroup of E. coli. A wide array of infections from mild and almost unapparent diarrhea to more serious manifestations such as hemorrhagic colitis (HC) and the development of a life-threatening syndrome known as hemolytic uremic syndrome (HUS) are caused by EHEC/STEC. Infants and children are the main affected patients, and although the incidence of infection varies in different regions, the impact and importance of EHEC/STEC infections in public health is immense, being the main cause of acute renal failure in children in many countries. The perspective of EHEC/STEC infections has been previously described,153153 Guth BEC, Picheth CF, Gomes TAT. Escherichia coli situation in Brazil. In: Torres AG, ed. Pathogenic Escherichia coli in Latin America. Sharjah, United Arab Emirates: Betham Science Publishers Ltd.; 2010:162-178.,154154 Majowicz SE, Scallan E, Jones-Bitton A, et al. Global incidence of human Shiga toxin-producing Escherichia coli infections and deaths: a systematic review and knowledge synthesis. Foodborne Pathog Dis. 2014;6:447-455. but a considerable amount of information has been obtained in more recent years related to the epidemiology, ecology and virulence properties of these bacteria.

E. coli O157:H7 serotype was the first to be linked to HC and HUS cases in the early 1980s, and has been since then responsible for numerous outbreaks and sporadic cases of severe diseases all over the world, therefore considered to be the prototype of this pathogenic group of bacteria.155155 Kaper JB, O'Brien AD. Overview and historical perspectives. Microbiol Spectr. 2014;2(2). EHEC-0028-2014. It is well known that hundreds of other E. coli serotypes can harbor the stx genes, but epidemiological studies carried out worldwide have proven that only some of them have been responsible for causing human diseases. Some serogroups including O26, O45, O103, O111, O121 and O145 can be highlighted among those most commonly related to human infections.156156 Gould LH, Mody RK, Ong KL, et al. Increased recognition of non-O157 Shiga toxin-producing Escherichia coli infections in the United States during 2000-2010: epidemiologic features and comparison with E. coli O157 infections. Food Pathog Dis. 2013;10:453-460. Moreover, in recent years the emergence of some particular clones such as the hybrid O104:H4 enteroaggregative E. coli carrying Stx2 genes, responsible for a severe outbreak of HUS starting in Germany in 2011,157157 Muniesa M, Hammerl JA, Stefan Hertwig S, Appel B, Brüssow H. Shiga toxin-producing Escherichia coli O104:H4: a new challenge for microbiology. Appl Env Microbiol. 2012;78:4065-4073. the spread of a new O26:H11 clone in Europe,158158 Bletz S, Bielaszewska M, Leopold SR, et al. Evolution of enterohemorrhagic Escherichia coli O26 based on single-nucleotide polymorphisms. Genome Biol Evol. 2013;5:1807-1816. and some other hybrid clones,159159 Rivas M, Chinen I, Guth BEC. Enterohemorrhagic (Shiga toxin-producing) Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:97-123. suggests that the mobility of genes and certainly the host background are important features implicated in their pathogenic potential.

Virulence factors, mechanisms and pathogenesis

The common feature among EHEC/STEC isolates is the ability to produce Stx. This family of toxins has a conserved AB5 subunit structure, composed of one active A subunit linked to a pentameric B subunit responsible for the binding of the toxin to specific glycolipid receptors on the surface of target cells. The stx operon is usually found within the sequence for an inducible, lysogenic, lambda-like bacteriophage. Stxs inhibit protein synthesis by removing an adenine residue from the 28S rRNA of the 60S ribosome.152152 Melton-Celsa AR. Shiga toxin (Stx) classification, structure, and function. Microbiol Spectr. 2014;2(3). EHEC-0024-2013. However, besides this activity, studies have described that Stx also acts on cell signal transduction and immune modulation causing proinflammatory and pro-apoptotic responses.159159 Rivas M, Chinen I, Guth BEC. Enterohemorrhagic (Shiga toxin-producing) Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:97-123. Two major families, Stx1 and Stx2, have been recognized, and on the basis of sequence diversity, each is composed of several variants. The Stx1 family is more homogenous and includes Stx1a, Stx1c and Stx1d; while the heterogenous Stx2 group is composed of Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g.160160 Scheutz F, Teel LD, Beutin L, et al. Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J Clin Microbiol. 2012;50:2951-2963. It should be mentioned that the association of some variants such as Stx2a, Stx2c or Stx2d with HC and HUS has been highlighted compared to some others that seemed to be more related to uncomplicated cases of diarrhea such as Stx1variants or even Stx2e, Stx2f and Stx2g, which are uncommonly found causing human infections so far.161161 Scheutz F. Taxonomy meets public health: the case of Shiga toxin-producing Escherichia coli. Microbiol Spectr. 2014;2(4). EHEC-0019-2013.,162162 Persad AK, LeJeune JT. Animal reservoirs of Shiga toxin-producing Escherichia coli. Microbiol Spectr. 2014;2(4). EHEC-0027-2014. Indeed, the higher association of Stx2 with severe diseases has been extensively studied by using Vero and endothelial cell lines as well as some animal models.159159 Rivas M, Chinen I, Guth BEC. Enterohemorrhagic (Shiga toxin-producing) Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:97-123. Moreover, knowledge of stx phage characteristics and behavior has helped our understanding of how differences in expression of Stx between EHEC/STEC isolates may contribute to pathogenesis and disease.163163 Krüger A, Lucchesi PMA. Shiga toxins and stx phages: highly diverse entities. Microbiology. 2015;161:451-462.

The ability to adhere to intestinal epithelial cells is another key event in EHEC/STEC pathogenesis. The presence of the chromosomal pathogenicity island LEE,164164 Stevens MP, Frankel GM. The locus of enterocyte effacement and associated virulence factors of enterohemorrhagic Escherichia coli. Microbiol Spectr. 2014;2(4). EHEC-0007-2013. also present in isolates belonging to the EPEC pathotype, is common. Although LEE has been described in the major EHEC/STEC serotypes responsible for a high proportion of HC and HUS cases in several countries, its presence is not a required condition for the occurrence of more serious infections as initially thought, because some LEE-negative strains are also capable of causing outbreaks and sporadic cases of HUS.165165 Paton AW, Woodrow MC, Doyle R, et al. Molecular characterization of a Shiga-toxigenic Escherichia coli O113:H21 strain lacking eae responsible for a cluster of cases of hemolytic-uremic syndrome. J Clin Microbiol. 1999;37:3357-3361.,166166 Bielaszewska M, Mellmann A, Zhang W, et al. Characterization of the E. coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study. Lancet Infect Dis. 2011;11:671-676.

Therefore, it is clear that EHEC/STEC pathogenesis is a multistep process, and besides the production of Stx toxins and the AE lesion, other factors including different types of toxins and adhesins have been described and found to be involved in virulence.159159 Rivas M, Chinen I, Guth BEC. Enterohemorrhagic (Shiga toxin-producing) Escherichia coli. In: Torres AG, ed. Escherichia coli in the Americas. Springer International Publishing; 2016:97-123.

One should also consider that as a pathogen of the human gastrointestinal tract the ability of EHEC/STEC to monitor nutrients in the gut milieu, and translate this information to sense the host physiological state in order to program the expression of its virulence markers has a pivotal role on the development of infection.167167 Pacheco A, Sperandio V. Enteric pathogens exploit the microbiota-generated nutritional environment of the gut. Microbiol Spectr. 2015;3(3), http://dx.doi.org/10.1128/microbiolspec.MBP-0001-2014.
http://dx.doi.org/10.1128/microbiolspec....
In addition, it has been shown that EHEC/STEC can also cross-communicate with the host by exploiting the autoinducer-3 (AI-3)/epinephrine/norepinephrine signaling system to express two important virulence traits, motility and A/E lesion, required at different time points during intestinal colonization.168168 Moreira CG, Sperandio V. The Epinephrine/norepinephrine/autoinducer-3 interkingdom signaling system in Escherichia coli O157:H7. Adv Exp Med Biol. 2016;874:247-261.

The ability to adhere, colonize and form biofilm on food and several types of surfaces may be a way to be an important source and/or vehicle of transmission of EHEC/STEC. In addition, biofilm may also act as bacterial protection against adverse environmental conditions. A study conducted by Biscola et al.,169169 Biscola FT, Abe CM, Guth BEC. Determination of adhesin gene sequences in, and biofilm formation by, O157 and non-O157 Shiga toxin-producing Escherichia coli strains isolated from different sources. Appl Environ Microbiol. 2011;77(7):2201-2208. evaluated the capacity of biofilm formation in EHEC/STEC strains isolated from different reservoirs and serotypes. The authors observed that the ability to adhere to abiotic surfaces forming biofilms, under defined culture conditions, occurred in an array of wild-type O157 and non-O157 strains. Biofilm production was identified in several non-O157 STEC serotypes of human, animal, and food origin. On the other hand, among the O157 strains, only those isolated from the animal reservoir and from a water sample produced biofilm. A close correlation between biofilm formation and expression of curli fimbriae and cellulose was observed among O157 strains. However, in addition to curli, the presence of other factors such as type 1 fimbriae and AT proteins may be associated with the ability to form biofilm in non-O157 strains. Matheus-Guimarães et al.,170170 Matheus-Guimarães C, Gonçalves E, Guth BEC. Interactions of O157 and non-O157 Shiga toxin-producing Escherichia coli (STEC) recovered from bovine hide and carcass with human cells and abiotic surfaces. Foodborne Pathog Dis. 2014;3:248-255. studied O157 and non-O157 EHEC/STEC strains isolated from bovine hides and carcasses and showed that different sets of genes were involved in the interactions of the bacteria with biotic and abiotic surfaces. Moreover, the detection of an O157 strain that was able to form biofilm on both glass and polystyrene and that adhered to and invaded human cells, suggests an important ability of this isolate to persist in the environment and interact with the host. In fact, cell invasion and survival of some EHEC/STEC strains in cultured human intestinal epithelial cells has been previously described.171171 Cordeiro F, Silva RIK, Vargas-Stampe TLZ, Cerqueira AMF, Andrade JRC. Cell invasion and survival of Shiga toxin-producing Escherichia coli within cultured human intestinal epithelial cells. Microbiol. 2013;159:1683-1694. It should be mentioned that this invasive characteristic has been identified in some EHEC/STEC serotypes, many of which are responsible for human infections.170170 Matheus-Guimarães C, Gonçalves E, Guth BEC. Interactions of O157 and non-O157 Shiga toxin-producing Escherichia coli (STEC) recovered from bovine hide and carcass with human cells and abiotic surfaces. Foodborne Pathog Dis. 2014;3:248-255.

171 Cordeiro F, Silva RIK, Vargas-Stampe TLZ, Cerqueira AMF, Andrade JRC. Cell invasion and survival of Shiga toxin-producing Escherichia coli within cultured human intestinal epithelial cells. Microbiol. 2013;159:1683-1694.

172 Dos Santos LF [PhD thesis] Studies on the Virulence Potential and Phylogeny of O113:H21 Escherichia coli Strains. Universidade Federal de São Paulo; 2011.
-173173 Gonzalez AG, Cerqueira AM, Guth BEC, et al. Serotypes, virulence markers and cell invasion ability of Shiga toxin-producing Escherichia coli (STEC) strains isolated from healthy dairy cattle. J Appl Microbiol. 2016;121:1130-1143. Therefore, it is conceivable that this virulence strategy may help bacteria to overcome host defense mechanisms and certainly contributes to their persistence in the zoonotic reservoir, ensuring efficient environmental and food transmission.

Another topic of interest has been the analysis and comparison of the virulence profile of EHEC/STEC strains isolated from the animal reservoir and environment with strains recovered from human infections. In general, these studies have shown that despite serotype diversity, the stx subtypes and the virulence profile identified among isolates from the animal reservoir and environment are similar to the isolates recovered from patients.173173 Gonzalez AG, Cerqueira AM, Guth BEC, et al. Serotypes, virulence markers and cell invasion ability of Shiga toxin-producing Escherichia coli (STEC) strains isolated from healthy dairy cattle. J Appl Microbiol. 2016;121:1130-1143.,178178 Lascowski KMS, Gonçalves EM, Alvares PP, et al. Prevalence and virulence profiles of Shiga toxin-producing Escherichia coli isolated from beef cattle in a Brazilian slaughterhouse. Zoon Publ Health. 2012;59(Suppl 1):19-90.

179 Beraldo LG, Borges CA, Maluta RP, Cardozo MV, Rigobelo EC, A’vila FA. Detection of Shiga toxigenic (STEC) and enteropathogenic (EPEC) Escherichia coli in dairy buffalo. Vet Microbiol. 2014;170:162-166.
-180180 Martins FH, Guth BEC, Piazza RM, et al. Diversity of Shiga toxin-producing Escherichia coli in sheep flocks of Paraná State, Southern Brazil. Vet Microbiol. 2015;175:150-156. There has been particular interest in some STEC serotypes that have been responsible for causing severe human infections, such as O113:H21, but unlike others, they do not produce adhesins encoded by LEE. By using a PCR microarray, 41 virulence or genetic markers were tested in a panel of 65 O113:H21 strains isolated from clinical infections, environment and food from various countries.174174 Feng PCH, Delannoy S, Lacher DW, et al. Genetic diversity and virulence potential of Shiga toxin-producing Escherichia coli O113:H21 strains isolated from clinical, environmental, and food sources. Appl Environ Microbiol. 2014;80:4757-4763. The results obtained showed no clear differences in these genetic markers between the pathogens recovered from HUS cases and the environmental strains. Moreover, only stx subtypes associated with human infections were identified in all isolates, therefore suggesting that the environmental isolates have the potential to cause human diseases.

Epidemiology

The incidence of HUS cases in Brazil is low,175175 De Souza RL, Carvalhaes JTA, Nishimura LS, Andrade MC, Guth BEC. Hemolytic uremic syndrome in pediatric intensive care units in São Paulo, Brazil. Open Microbiol J. 2011;5:76-82. and although some hypothesis has been proposed to explain this fact, there are limited data on the immune response against Stx. In an attempt to overcome this gap, prevalence of anti-Stx2 antibodies in sera of children diagnosed with HUS and of healthy children was recently determined.176176 Guirro M, Piazza RMF, de Souza RL, Guth BEC. Humoral immune response to Shiga Toxin 2 (Stx2) among Brazilian urban children with hemolytic uremic syndrome and healthy controls. BMC Infect Dis. 2014;14:320-325. The percentage of individuals showing antibodies against Stx2 was higher among HUS patients than controls, and the results also confirmed that STEC strains are circulating in our settings despite the low number of identified HUS cases.

Among the several serotypes associated with human infections, O157:H7 is responsible for more severe cases. Epidemiological investigations of diarrheal outbreaks conducted in four Brazilian states showed that O157:H7 strains were isolated from two hospitalized patients, one with HUS and the other with bloody diarrhea.177177 Dos Santos LF, Guth BEC, Hernandes RT, et al. Shiga toxin-producing Escherichia coli in Brazil: human infections from 2007 to 2014. In: 9th Triennial International Symposium on Shiga Toxin (Verocytotoxin)-producing Escherichia coli (VTEC), Boston, vol. 87. 2015. Besides, O157:H7, EHEC/STEC strains belonging to the top six most important non-O157 serogroups such as O26, O103, O111 and O145 were identified, all of which were recovered from ambulatory patients. In addition, some uncommon serogroups including O1, O24 and O77 among others were also detected, but they were all associated with acute diarrhea. It is interesting to note that the majority of patients from whom STEC was isolated were female (57%), and that patients’ ages ranged from 8 months to 80 years, with most being less than five years old (54%).177177 Dos Santos LF, Guth BEC, Hernandes RT, et al. Shiga toxin-producing Escherichia coli in Brazil: human infections from 2007 to 2014. In: 9th Triennial International Symposium on Shiga Toxin (Verocytotoxin)-producing Escherichia coli (VTEC), Boston, vol. 87. 2015.

The distribution of EHEC/STEC in the gastrointestinal tract of a wide variety of animals indicates the zoonotic character of its infections. The role of different animal species as asymptomatic carriers of EHEC/STEC has been extensively studied in the last years in Brazil. Besides cattle, which are their most common natural reservoir,173173 Gonzalez AG, Cerqueira AM, Guth BEC, et al. Serotypes, virulence markers and cell invasion ability of Shiga toxin-producing Escherichia coli (STEC) strains isolated from healthy dairy cattle. J Appl Microbiol. 2016;121:1130-1143.,178178 Lascowski KMS, Gonçalves EM, Alvares PP, et al. Prevalence and virulence profiles of Shiga toxin-producing Escherichia coli isolated from beef cattle in a Brazilian slaughterhouse. Zoon Publ Health. 2012;59(Suppl 1):19-90. the presence of these pathogens has been identified in the feces of dairy buffaloes,179179 Beraldo LG, Borges CA, Maluta RP, Cardozo MV, Rigobelo EC, A’vila FA. Detection of Shiga toxigenic (STEC) and enteropathogenic (EPEC) Escherichia coli in dairy buffalo. Vet Microbiol. 2014;170:162-166. sheep,180180 Martins FH, Guth BEC, Piazza RM, et al. Diversity of Shiga toxin-producing Escherichia coli in sheep flocks of Paraná State, Southern Brazil. Vet Microbiol. 2015;175:150-156.,181181 Maluta RP, Fairbrother JM, Stella AE, Rigobelo EC, Martinez R, A’vila FA. Potentially pathogenic Escherichia coli in healthy, pasture-raised sheep on farms and at the abattoir in Brazil. Vet Microbiol. 2014;169:89-95. pigs,182182 Borges CA, Beraldo LG, Maluta RP, et al. Shiga toxigenic and atypical enteropathogenic Escherichia coli in the feces and carcasses of slaughtered pigs. Foodborne Pathog Dis. 2012;10:1-7.,183183 Martins RP, Silva MC, Dutra V, Nakazato L, Leite DS. Preliminary virulence genotyping and phylogeny of Escherichia coli from the gut of pigs at slaughtering stage in Brazil. Meat Sci. 2013;93:437-440. birds,184184 Gioia-Di Chiacchio RM, Cunha MPV, Sturn RM, et al. Shiga toxin-producing Escherichia coli (STEC): zoonotic risks associated with psittacine pet birds in home environments. Vet Microbiol. 2016;184:27-30. and fishes.185185 Ribeiro LF, Barbosa MMC, Pinto FR. Shiga toxigenic and enteropathogenic Escherichia coli in water and fish from pay-to-fish ponds. Lett Appl Microbiol. 2015;62:216-220. It is noteworthy that some relevant serotypes linked to human infections such as O103:H2 and O157:H7 have been recovered from the feces of sheep186186 Martins FH, Guth BEC, Piazza RMF, Blanco J, Pelayo JS. First description of a Shiga toxin-producing Escherichia coli O103:H2 strain isolated from sheep in Brazil. J Infect Dev Ctries. 2014;8:126-128. and cattle173173 Gonzalez AG, Cerqueira AM, Guth BEC, et al. Serotypes, virulence markers and cell invasion ability of Shiga toxin-producing Escherichia coli (STEC) strains isolated from healthy dairy cattle. J Appl Microbiol. 2016;121:1130-1143. respectively. Additionally, the high prevalence of O157:H7 EHEC/STEC strains identified in hides of cattle sent to slaughter in a Brazilian processing plant178178 Lascowski KMS, Gonçalves EM, Alvares PP, et al. Prevalence and virulence profiles of Shiga toxin-producing Escherichia coli isolated from beef cattle in a Brazilian slaughterhouse. Zoon Publ Health. 2012;59(Suppl 1):19-90. certainly represents a relevant issue that should be considered when thinking about interventions targeting EHEC/STEC related to animal handling, from farm to slaughter, as well as the implementation of food safety throughout production and processing.

The presence of EHEC/STEC in the environment is another issue of concern, since they can survive in the soil, manure, pastures and water, which thus represent important vehicles of transmission. The isolation of STEC strains from drinking water supplies, collected in different municipalities in northern Paraná State, has been recently described, highlighting the importance of drinking water, especially that from untreated water supplies, as a source of STEC strains potentially pathogenic for humans.187187 Lascowski KMS, Guth BEC, Martins FH, Rocha SPD, Irino K, Pelayo JS. Shiga toxin-producing Escherichia coli in drinking water supplies of North Paraná State, Brazil. J Appl Microbiol. 2013;114:1230-1239. Taking into account that chicken litter is very useful as an organic soil fertilizer for the production of fruits and vegetables in our settings, the detection of STEC in organic chicken fertilizer used on farms188188 Puño-Sarmiento J, Gazal LE, Medeiros LP, Nishio EK, Kobayashi RKT, Nakazato G. Identification of diarrheagenic Escherichia coli strains from avian organic fertilizers. Int J Environ Res Public Health. 2014;11:8924-8939. also represents a significant public health safety hazard.

Although data on the detection of EHEC/STEC in foods in Brazil are still scarce, the isolation and identification of O157:H7 serotype from a ground beef sample was described for the first time,189189 Lucatelli A, Ms Thesis Shiga Toxin-producing Escherichia coli in Ground Beef at Retail Level at São Paulo City, Brazil. Faculdade de Ciências Farmacêuticas, Universidade de São Paulo; 2012. while O125:H19 and O149:H8 STEC serotypes were found in refrigerated raw kibbe collected from retail establishments.190190 Peresi JTM, Almeida IAZC, Vaz TMI, et al. Search for diarrheagenic Escherichia coli in raw kibbe samples reveals the presence of Shiga toxin-producing strains. Food Control. 2016;63:165-170. On the other hand, EHEC/STEC has not been detected in pasteurized cow's milk samples collected in dairies in northwestern Paraná State191191 Hoffmann SA, Pieretti GG, Fiorini A, Patussi EV, Cardoso RF, Mikcha JMG. Shiga-toxin genes and genetic diversity of Escherichia coli isolated from pasteurized cow milk in Brazil. J Food Sci. 2014;79(6):1175-1180. or in raw milk, pasteurized milk, Minas Frescal cheese and ground beef samples collected in Minas Gerais.192192 Leite Junior BRC, Oliveira PM, Silva FJM. Occurrence of Shiga toxin-producing Escherichia coli (STEC) in bovine feces, feed, water, raw milk, pasteurized milk, Minas Frescal cheese and ground beef samples collected in Minas Gerais, Brazil. Int Food Res J. 2014;21(6):2481-2486. One should be aware that despite difficulties in the detection and isolation of EHEC/STEC from foods, the implementation of the most sensitive methods in most laboratories should be the main goal in the near future to help in the analysis of the risk posed by foods as vehicles of STEC transmission to humans.

Detection and diagnosis

An important concern is how to detect Shiga toxin-producing strains either in stools of infected patients or contaminated food, since selective enrichment is necessary.193193 Chapman PA, Siddons CA. A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli 0157 from cases of bloody diarrhoea, non-bloody diarrhoea and asymptomatic contacts. J Med Microbiol. 1996;44:267-271.,194194 Bopp CA, Brenner FW, Fields PI, Wells JG, Strockbine NA. Escherichia, Shigella, and Salmonella. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, eds. Manual of Clinical Microbiology. 8th edition Washington, DC: ASM Press; 2003. For routine diagnosis, some protocols have already been described.139139 Piazza RMF, Abe CM, Horton DSPQ, et al. Detection and subtyping methods of diarrheagenic Escherichia coli strains. In: Torres AG, ed. Pathogenic Escherichia coli in Latin America. Bentham Science Publishers; 2010:95-115. However, the gold standard for Stx detection is still the evaluation of the cytotoxicity of bacterial culture supernatants to eukaryotic cells.195195 Konowalchuk J, Speirs JI, Stavric S. Vero response to a cytotoxin of Escherichia coli. Infect Immun. 1977;18:775-779.,196196 Karmali MA, Steele BT, Petric M, Lim C. Sporadic cases of hemolytic uremic syndrome associated with fecal cytotoxin and cytotoxin-producing Escherichia coli. Lancet. 1983;1(8325):619-620. Thus, multiplex PCR including stx gene and other virulence genes could be useful in screening for STEC using bacterial confluent growth zones or sorbitol fermenting and non-fermenting colonies taken from SMAC.197197 Leotta GA, Chinen I, Epszteyn S, et al. Validation of a multiplex PCR for detection of Shiga toxin-producing Escherichia coli. Rev Argent Microbiol. 2005;37:1-10.

Numerous assays for the diagnosis of STEC have been developed on the basis of the detection of Stx1 and/or Stx2, which represents the major virulence factors of this E. coli category.198198 Center of Disease Control of United States, Centers for Disease Control and Prevention. Recommendations for diagnosis of Shiga toxin-producing Escherichia coli infections by clinical laboratories. MMWR. 2009;58:1-12. Sensitivities and specificities vary according to the test format and the manufacturer.199199 Donohue-Rolfe A, Kelley MA, Bennish M, Keush GT. Enzyme-linked immunosorbent assay for Shigella toxin. J Clin Microbiol. 1986;24:65-68.

200 Kongmuang U, Honda T, Miwatani T. Enzyme-linked immunosorbent assay to detect Shiga toxin of Shigella dysenteriae and related toxins. J Clin Microbiol. 1987;25:115-118.

201 Mackenzie AMR, Lebel P, Orrbine E, et al. Sensitivities and specificities of premier E. coli O157 and premier EHEC enzyme immunoassays for diagnosis of infection with verotoxin (Shiga-like toxin) producing Escherichia coli. J Clin Microbiol. 1998;36:160811.

202 Novick TJ, Daly JA, Mottice SL, Carroll KC. Comparison of sorbitol MacConkey agar and a two-step method which utilizes enzyme-linked immunosorbent assay toxin testing and a chromogenic agar to detect and isolate enterohemorrhagic Escherichia coli. J Clin Microbiol. 2000;38:547-551.

203 Beutin L, Zimmermann S, Gleier K. Rapid detection and isolation of Shiga-like toxin (verocytotoxin)-producing Escherichia coli by direct testing of individual enterohemolytic colonies from washed sheep blood agar plates in the VTEC-RPLA assay. Clin Microbiol. 1996;34:2812-2814.

204 Beutin L, Zimmermann S, Gleier K. Evaluation of the VTEC-Screen “Seiken” test for detection of different types of Shiga toxin (verotoxin)-producing Escherichia coli (STEC) in human stool samples. Diagn Microbiol Infect Dis. 2002;42:1-8.
-205205 Beutin L, Steinrück H, Krause G, et al. Comparative evaluation of the Ridascreen® Verotoxin enzyme immunoassay for detection of Shiga-toxin producing strains of Escherichia coli (STEC) from food and other sources. J Appl Microbiol. 2007;102:630-639. Nevertheless, the standard by which each manufacturer evaluates its tests also varies; therefore, a direct comparison of performance characteristics of various immunoassays has not been performed.198198 Center of Disease Control of United States, Centers for Disease Control and Prevention. Recommendations for diagnosis of Shiga toxin-producing Escherichia coli infections by clinical laboratories. MMWR. 2009;58:1-12.,206206 Gould LH, Bopp C, Strockbine N, et al. Recommendations for diagnosis of Shiga toxin-producing Escherichia coli infections by clinical laboratories. MMWR Recomm Rep. 2009;58:1-14.,207207 Rocha LB, Luz D, Moraes CTP, et al. Interaction between Shiga toxin and monoclonal antibodies: binding characteristics and in vitro neutralizing abilities. Toxins. 2012;4:729-747. Moreover, these commercially available tests are not affordable for developing countries. Thus, to outline this, previous works have established different formats of immunoassays, employing either a mixture of rabbit anti-Stx1 and anti-Stx2 sera by indirect ELISA or polyclonal and monoclonal antibodies in a capture ELISA assay for the detection of STEC.207207 Rocha LB, Luz D, Moraes CTP, et al. Interaction between Shiga toxin and monoclonal antibodies: binding characteristics and in vitro neutralizing abilities. Toxins. 2012;4:729-747.

208 Rocha LB, Piazza RMF. Production of Shiga toxin by Shiga toxin-expressing Escherichia coli (STEC) in broth media: from divergence to definition. Lett Appl Microbiol. 2007;45:411-417.
-209209 Mendes-Ledesma MRB, Rocha LB, Bueris V, et al. Production and characterization of rabbit polyclonal sera against Shiga toxins stx1 and stx2 for detection of Shiga toxin producing. Microbiol Immunol. 2008;52:484-491. The standardized methods are reproducible, fast, easy to perform, showing high sensitivity in detecting Stx by capture ELISA, even in low-producing isolates. These assays have not yet been evaluated in terms of industrial quality control and commercial availability, but the estimated cost of the assay is around US$70 per 96 detections, which is realistically inexpensive for developing countries.

These monoclonal antibodies were rebuilt resulting in single chain fragment variable (scFv) fragments. Stx2-scFv was obtained from a bacteria-induced culture and showed diagnostic ability; the scFv fragment was able to recognize the majority of Stx2-producing strains, with 79.3% sensitivity (confidence interval of 60.3 to 92%), and no reactivity was observed with the non-producing strains, indicating as high as 100% specificity (confidence interval of 86.8-100%).210210 Luz D, Chen H, Maranhão AQ, Rocha LB, Sidhu S, Piazza RMF. Development and characterization of recombinant antibody fragments that recognize and neutralize in vitro stx2 toxin from Shiga toxin-producing Escherichia coli. PLoS ONE. 2015;10(3):0120481. It is worth mentioning that none of the commercially available immunoenzymatic tests for Stx1/2 toxin detection employ recombinant antibodies produced in bacteria, which indeed will reduce the costs of the diagnostic assays.198198 Center of Disease Control of United States, Centers for Disease Control and Prevention. Recommendations for diagnosis of Shiga toxin-producing Escherichia coli infections by clinical laboratories. MMWR. 2009;58:1-12.

Enteroaggregative E. coli

EAEC is the diarrheagenic E. coli pathotype defined by showing the characteristic AA pattern on epithelial cells in culture.211211 Navarro-Garcia F, Elias WP. Autotransporters and virulence of enteroaggregative E. coli. Gut Microbes. 2011;2:13-24. The AA pattern was defined in 1987 when Nataro et al.,212212 Nataro JP, Kaper JB, Robins-Browne R, Prado V, Vial P, Levine MM. Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells. Pediatr Infect Dis J. 1987;6:829-831. distinguished the previously described “diffuse adherence” as the truly diffuse adherence (DA) and the AA pattern. The standard AA was characterized by adherent bacteria in a stacked-brick arrangement on the surface of epithelial cells and also on the coverslip between cells. Strains displaying the AA pattern were then categorized as “enteroadherent-aggregative E. coli” but afterwards the category was called enteroaggregative E. coli or EAEC, the current nomenclature. The detection of AA in vitro is still the gold standard test to define EAEC; however, as described before, the AA pattern may be found in strains of other DEC pathotypes, such as aEPEC. Therefore, an up-to-date definition of EAEC is the diarrheagenic E. coli that produce AA in cultured epithelial cells but lack the main genetic markers that define other DEC pathotypes (EPEC, ETEC, EHEC, EIEC). An exception for that is the hybrid EAEC/STEC strain responsible for a massive outbreak of diarrhea and HUS in 2011 in Europe.213213 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. This strain consists of an EAEC strain that acquired the Stx2-encoding phage. Therefore, this specific O104:H4 strain is a Stx-producing EAEC.

Diarrhea caused by EAEC is watery, often with the presence of mucus, with or without blood and abdominal pain, vomiting and low fever. Acute self-limiting diarrhea is the usual pathology, but some patients may develop protracted diarrhea, i.e., lasting more than 14 days.214214 Hebbelstrup Jensen B, Olsen KE, Struve C, Krogfelt KA, Petersen AM. Epidemiology and clinical manifestations of enteroaggregative Escherichia coli. Clin Microbiol Rev. 2014;27:614-630. Prolonged diarrhea occurs depending on the host's immunity, nutritional status and genetic susceptibility.215215 Lima AA, Guerrant RL. Persistent diarrhea in children: epidemiology, risk factors, pathophysiology, nutritional impact, and management. Epidemiol Rev. 1992;14:222-242. Genetic susceptibilities associated with EAEC diarrhea were identified in North American travelers to Mexico. Single nucleotide polymorphisms (SNP) in the IL-8 gene promoter and the promotor regions of the genes encoding lactoferrin, CD14 and osteoprotegerin as well were recognized as indicators for symptomatic EAEC infection.216216 Jiang ZD, Okhuysen PC, Guo DC, et al. Genetic susceptibility to enteroaggregative Escherichia coli diarrhea: polymorphism in the interleukin-8 promotor region. J Infect Dis. 2003;188:506-511.

217 Mohamed JA, DuPont HL, Jiang ZD, et al. A novel single-nucleotide polymorphism in the lactoferrin gene is associated with susceptibility to diarrhea in North American travelers to Mexico. Clin Infect Dis. 2007;44:945-952.

218 Mohamed JA, DuPont HL, Jiang ZD, et al. A single-nucleotide polymorphism in the gene encoding osteoprotegerin, an anti-inflammatory protein produced in response to infection with diarrheagenic Escherichia coli, is associated with an increased risk of nonsecretory bacterial diarrhea in North American travelers to Mexico. J Infect Dis. 2009;199:477-485.
-219219 Mohamed JA, DuPont HL, Flores J, et al. Single nucleotide polymorphisms in the promoter of the gene encoding the lipopolysaccharide receptor CD14 are associated with bacterial diarrhea in US and Canadian travelers to Mexico. Clin Infect Dis. 2011;52:1332-1341.

A well-described characteristic of EAEC strains is their heterogeneous nature when serotypes, genetic markers of virulence and phylogenetic groups are analyzed.220220 Czeczulin JR, Whittam TS, Henderson IR, Navarro-Garcia F, Nataro JP. Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli. Infect Immun. 1999;67:2692-2699.

221 Suzart S, Guth BE, Pedroso MZ, Okafor UM, Gomes TA. Diversity of surface structures and virulence genetic markers among enteroaggregative Escherichia coli (EAEC) strains with and without the EAEC DNA probe sequence. FEMS Microbiol Lett. 2001;201:163-168.

222 Elias WP, Uber AP, Tomita SK, Trabulsi LR, Gomes TA. Combinations of putative virulence markers in typical and variant enteroaggregative Escherichia coli strains from children with and without diarrhoea. Epidemiol Infect. 2002;129:49-55.

223 Boisen N, Scheutz F, Rasko DA, et al. Genomic characterization of enteroaggregative Escherichia coli from children in Mali. J Infect Dis. 2012;205:431-444.

224 Chattaway MA, Jenkins C, Rajendram D, et al. Enteroaggregative Escherichia coli have evolved independently as distinct complexes within the E. coli population with varying ability to cause disease. PLoS ONE. 2014;9(11):e112967.
-225225 Okeke IN, Wallace-Gadsden F, Simons HR, et al. Multi-locus sequence typing of enteroaggregative Escherichia coli isolates from Nigerian children uncovers multiple lineages. PLoS ONE. 2010;5(11):e14093. This indicates that only EAEC strains carrying specific virulence factors are able to cause diarrhea. While these factors are unknown, some studies have demonstrated the association of specific virulence genes with diarrhea, such as pet or aafA in Brazil226226 Lima IF, Boisen N, Quetz Jda S, et al. Prevalence of enteroaggregative Escherichia coli and its virulence-related genes in a case-control study among children from north-eastern Brazil. J Med Microbiol. 2013;62:683-693. and sepA in Mali.223223 Boisen N, Scheutz F, Rasko DA, et al. Genomic characterization of enteroaggregative Escherichia coli from children in Mali. J Infect Dis. 2012;205:431-444.

Virulence factors, mechanisms and pathogenesis

Most of our knowledge about EAEC pathogenesis is based on data accumulated from studies with EAEC strain 042, since its association with human diarrhea in a volunteer study.227227 Nataro JP, Deng Y, Cookson S, et al. Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers. J Infect Dis. 1995;171:465-468. These putative virulence factors include adhesins, toxins and secreted proteins. However, none of these factors are found in all EAEC strains.

The majority of these virulence factors are plasmid borne, including those mediating AA. Consequently, these high-molecular-weight plasmids are called pAA.220220 Czeczulin JR, Whittam TS, Henderson IR, Navarro-Garcia F, Nataro JP. Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli. Infect Immun. 1999;67:2692-2699. Baudry et al.,228228 Baudry B, Savarino SJ, Vial P, Kaper JB, Levine MM. A sensitive and specific DNA probe to identify enteroaggregative Escherichia coli, a recently discovered diarrheal pathogen. J Infect Dis. 1990;161:1249-1251. developed a genetic probe (CVD432) for EAEC diagnosis on the basis of a fragment from pAA1 present in EAEC strain 17-2. In EAEC 042, many putative virulence factors are present in pAA2.220220 Czeczulin JR, Whittam TS, Henderson IR, Navarro-Garcia F, Nataro JP. Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli. Infect Immun. 1999;67:2692-2699.

Recently, a division of EAEC strains into typical or atypical subgroups was proposed. This classification is based on the presence or absence of aggR, a gene that encodes a global regulator of EAEC virulence genes.229229 Sarantuya J, Nishi J, Wakimoto N, et al. Typical enteroaggregative Escherichia coli is the most prevalent pathotype among E. coli strains causing diarrhea in Mongolian children. J Clin Microbiol. 2004;42:133-139. Therefore, it has been proposed that typical EAEC have more pathogenic potential by the presence of the AggR regulon and, consequently, pAA virulence factors.230230 Morin N, Santiago AE, Ernst RK, Guillot SJ, Nataro JP. Characterization of the AggR regulon in enteroaggregative Escherichia coli. Infect Immun. 2013;81:122-132. However, at least two outbreaks of diarrhea were caused by atypical EAEC,231231 Cobeljic M, Miljkovic-Selimovic B, Paunovic-Todosijevic D, et al. Enteroaggregative Escherichia coli associated with an outbreak of diarrhoea in a neonatal nursery ward. Epidemiol Infect. 1996;117:11-16.,232232 Itoh Y, Nagano I, Kunishima M, Ezaki T. Laboratory investigation of enteroaggregative Escherichia coli O untypeable:H10 associated with a massive outbreak of gastrointestinal illness. J Clin Microbiol. 1997;35:2546-2550. and atypical EAEC are commonly isolated from children with diarrhea, in some cases more frequently than typical strains.233233 Huang DB, Jiang ZD, Dupont HL. Association of virulence factor-positive and -negative enteroaggregative Escherichia coli and occurrence of clinical illness in travelers from the United States to Mexico. Am J Trop Med Hyg. 2003;69:506-508.,234234 Huang DB, Mohamed JA, Nataro JP, DuPont HL, Jiang ZD, Okhuysen PC. Virulence characteristics and the molecular epidemiology of enteroaggregative Escherichia coli isolates from travellers to developing countries. J Med Microbiol. 2007;56:1386-1392.

Numerous adhesins, cytotoxins, enterotoxins and secreted proteins have been characterized in EAEC strains since this pathotype definition.211211 Navarro-Garcia F, Elias WP. Autotransporters and virulence of enteroaggregative E. coli. Gut Microbes. 2011;2:13-24.,214214 Hebbelstrup Jensen B, Olsen KE, Struve C, Krogfelt KA, Petersen AM. Epidemiology and clinical manifestations of enteroaggregative Escherichia coli. Clin Microbiol Rev. 2014;27:614-630.

The most studied adhesins are the aggregative adherence fimbria (AAF/I-AAF/V) family, which includes five types.235235 Nataro JP, Deng Y, Maneval DR, German AL, Martin WC, Levine MM. Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes. Infect Immun. 1992;60:2297-2304.

236 Elias WP, Czeczulin JR, Henderson IR, Trabulsi LR, Nataro JP. Organization of biogenesis genes for aggregative adherence fimbria II defines a virulence gene cluster in enteroaggregative Escherichia coli. J Bacteriol. 1999;181:1779-1785.

237 Bernier C, Gounon P, Le Bouguenec C. Identification of an aggregative adhesion fimbria (AAF) type III-encoding operon in enteroaggregative Escherichia coli as a sensitive probe for detecting the AAF-encoding operon family. Infect Immun. 2002;70:4302-4311.

238 Boisen N, Struve C, Scheutz F, Krogfelt KA, Nataro JP. New adhesin of enteroaggregative Escherichia coli related to the Afa/Dr/AAF family. Infect Immun. 2008;76:3281-3292.
-239239 Jonsson R, Struve C, Boisen N, et al. Novel aggregative adherence fimbria variant of enteroaggregative Escherichia coli. Infect Immun. 2015;83:1396-1405. They mediate the AA pattern and biofilm formation. Afimbrial adhesins have also been characterized in EAEC strains, including outer membrane proteins between 30 and 58 kDa.240240 Suzart S, Gomes TAT, Guth BE. Characterization of serotypes and outer membrane protein profiles in enteroaggregative Escherichia coli strains. Microbiol Immunol. 1999;43:201-205.

241 Debroy C, Yealy J, Wilson RA, Bhan MK, Kumar R. Antibodies raised against the outer membrane protein interrupt adherence of enteroaggregative Escherichia coli. Infect Immun. 1995;63:2873-2879.
-242242 Monteiro-Neto V, Bando SY, Moreira-Filho CA, Giron JA. Characterization of an outer membrane protein associated with haemagglutination and adhesive properties of enteroaggregative Escherichia coli O111:H12. Cell Microbiol. 2003;5:533-547. However, it has been shown that these structures are present in low frequencies in EAEC collections from different settings.221221 Suzart S, Guth BE, Pedroso MZ, Okafor UM, Gomes TA. Diversity of surface structures and virulence genetic markers among enteroaggregative Escherichia coli (EAEC) strains with and without the EAEC DNA probe sequence. FEMS Microbiol Lett. 2001;201:163-168.,226226 Lima IF, Boisen N, Quetz Jda S, et al. Prevalence of enteroaggregative Escherichia coli and its virulence-related genes in a case-control study among children from north-eastern Brazil. J Med Microbiol. 2013;62:683-693.,243243 Zamboni A, Fabbricotti SH, Fagundes-Neto U, Scaletsky IC. Enteroaggregative Escherichia coli virulence factors are found to be associated with infantile diarrhea in Brazil. J Clin Microbiol. 2004;42:1058-1063.

244 Pereira AL, Ferraz LR, Silva RS, Giugliano LG. Enteroaggregative Escherichia coli virulence markers: positive association with distinct clinical characteristics and segregation into 3 enteropathogenic E. coli serogroups. J Infect Dis. 2007;195:366-374.
-245245 Regua-Mangia AH, Gomes TA, Vieira MA, Irino K, Teixeira LM. Molecular typing and virulence of enteroaggregative Escherichia coli strains isolated from children with and without diarrhoea in Rio de Janeiro city, Brazil. J Med Microbiol. 2009;58:414-422.

Located in pAA2 of EAEC 042 is the aap gene, encoding an antiaggregation protein called dispersin.246246 Sheikh J, Czeczulin JR, Harrington S, et al. A novel dispersin protein in enteroaggregative Escherichia coli. J Clin Invest. 2002;110:1329-1337. This protein is secreted and linked to lipopolysaccharide, neutralizing the negative charge of the bacterial surface leading to AAF projection and consequent dispersion along the intestinal mucosa.247247 Nishi J, Sheikh J, Mizuguchi K, et al. The export of coat protein from enteroaggregative Escherichia coli by a specific ATP-binding cassette transporter system. J Biol Chem. 2003;278:45680-45689. Although immunogenic, dispersin is found in other E. coli pathotypes and in commensal E. coli.248248 Monteiro BT, Campos LC, Sircili MP, et al. The dispersin-encoding gene (aap) is not restricted to enteroaggregative Escherichia coli. Diagn Microbiol Infect Dis. 2009;65:81-84.

Various toxins have been described in EAEC in association with the cytotoxic or enterotoxic effects of culture supernatants in vitro. The heat-stable toxin enteroaggregative E. coli heat-stable enterotoxin 1 (EAST-1) was the first toxin characterized in the EAEC pathotype.249249 Savarino SJ, Fasano A, Robertson DC, Levine MM. Enteroaggregative Escherichia coli elaborate a heat-stable enterotoxin demonstrable in an in vitro rabbit intestinal model. J Clin Invest. 1991;87:1450-1455. EAST-1 activates adenylate cyclase inducing increased cyclic GMP levels, effects observed in a Ussing chamber with rabbit ileum.250250 Savarino SJ, Fasano A, Watson J, et al. Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin. Proc Natl Acad Sci U S A. 1993;90:3093-3097. ShET1 is an A:B type toxin that causes accumulation of fluid in rabbit ileal loops and has secretory response in Ussing chamber assays.251251 Fasano A, Noriega FR, Liao FM, Wang W, Levine MM. Effect of Shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo. Gut. 1997;40:505-511.,252252 Menard LP, Lussier JG, Lepine F, Paiva de Sousa C, Dubreuil JD. Expression, purification, and biochemical characterization of enteroaggregative Escherichia coli heat-stable enterotoxin 1. Protein Expr Purif. 2004;33:223-231.

The two AT proteins characterized in EAEC 042, Pet and Pic,253253 Eslava C, Navarro-Garcia F, Czeczulin JR, Henderson IR, Cravioto A, Nataro JP. Pet, an autotransporter enterotoxin from enteroaggregative Escherichia coli. Infect Immun. 1998;66:3155-3163.-254254 Henderson IR, Czeczulin J, Eslava C, Noriega F, Nataro JP. Characterization of Pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli. Infect Immun. 1999;67:5587-5596. are members of the serine protease autotransporters of Enterobacteriaceae, or SPATE.255255 Dautin N. Serine protease autotransporters of enterobacteriaceae (SPATEs): biogenesis and function. Toxins. 2010;2:1179-1206. Pet is a cytotoxin that modifies the cytoskeleton of enterocytes, leading to rounding and cell detachment. The cytotoxic mechanism of Pet arises from the degradation of α-fodrin, a membrane protein of the enterocytes.256256 Navarro-Garcia F, Eslava C, Villaseca JM, et al. In vitro effects of a high-molecular-weight heat-labile enterotoxin from enteroaggregative Escherichia coli. Infect Immun. 1998;66:3149-3154. Pic is a multitask protein that mediates hemagglutination, mucus cleavage and hypersecretion, intestinal colonization in mice, cleavage of surface glycoproteins involved in leukocyte trafficking and cleavage of key complement molecules.257257 Ruiz-Perez F, Nataro JP. Bacterial serine proteases secreted by the autotransporter pathway: classification, specificity, and role in virulence. Cell Mol Life Sci. 2014;71:745-770.,258258 Abreu AG, Fraga TR, Granados Martínez AP, et al. The serine protease Pic from enteroaggregative Escherichia coli mediates immune evasion by the direct cleavage of complement proteins. J Infect Dis. 2015;212:106-115. The phenotypes identified for Pic suggest its role in promoting colonization of the intestine and immune system evasion. SPATEs are immunogenic proteins, as evidenced by the presence of serum antibodies against Pet and Pic in children recovering from diarrhea caused by EAEC.259259 Bellini EM, Elias WP, Gomes TA, et al. Antibody response against plasmid-encoded toxin (Pet) and the protein involved in intestinal colonization (Pic) in children with diarrhea produced by enteroaggregative Escherichia coli. FEMS Immunol Med Microbiol. 2005;43:259-264.

In the years that followed the definition of EAEC as a pathotype, research in the field was dedicated to prove the pathogenic capacity of EAEC using different animal models260260 Vial PA, Robins-Browne R, Lior H, et al. Characterization of enteroadherent-aggregative Escherichia coli, a putative agent of diarrheal disease. J Infect Dis. 1988;158:70-79.

261 Tickoo SK, Bhan MK, Srivastava R, et al. Intestinal colonization and production of diarrhoea by enteroadherent-aggregative Escherichia coli. Indian J Med Res. 1992;95:278-283.
-262262 Tzipori S, Montanaro J, Robins-Browne RM, Vial P, Gibson R, Levine MM. Studies with enteroaggregative Escherichia coli in the gnotobiotic piglet gastroenteritis model. Infect Immun. 1992;60:5302-5306. and human volunteers receiving oral inoculum of different EAEC strains.227227 Nataro JP, Deng Y, Cookson S, et al. Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers. J Infect Dis. 1995;171:465-468.,235235 Nataro JP, Deng Y, Maneval DR, German AL, Martin WC, Levine MM. Aggregative adherence fimbriae I of enteroaggregative Escherichia coli mediate adherence to HEp-2 cells and hemagglutination of human erythrocytes. Infect Immun. 1992;60:2297-2304.,263263 Mathewson JJ, Johnson PC, DuPont HL, Satterwhite TK, Winsor DK. Pathogenicity of enteroadherent Escherichia coli in adult volunteers. J Infect Dis. 1986;154:524-527. Not all volunteers developed diarrhea after ingestion of different EAEC strains, the first evidence that strains of this pathotype are heterogeneous. Among the strains tested, EAEC 042 (serotype O44:H18) caused diarrhea in three out of five volunteers.227227 Nataro JP, Deng Y, Cookson S, et al. Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers. J Infect Dis. 1995;171:465-468. Since then, strain 042 has been considered the prototype EAEC strain and is certainly the most studied strain of the pathotype.264264 Chaudhuri RR, Sebaihia M, Hobman JL, et al. Complete genome sequence and comparative metabolic profiling of the prototypical enteroaggregative Escherichia coli strain 042. PLoS ONE. 2010;5(1):e8801. EAEC 042 was isolated from a case of acute infantile diarrhea in Peru.265265 Nataro JP, Baldini MM, Kaper JB, Black RE, Bravo N, Levine MM. Detection of an adherence factor of enteropathogenic Escherichia coli with a DNA probe. J Infect Dis. 1985;152:560-565. The clinical data obtained from the volunteers who developed diarrhea suggested that EAEC 042 caused secretory diarrhea, with abundant presence of mucus and absence of blood in the stool.

Studies employing different EAEC strains interacting with intestinal cells from animals or humans have been performed to elucidate the pathogenesis of this pathotype. Data from these in vitro, in vivo and ex vivo experiments strongly indicate that EAEC can bind to jejunal, ileal and colonic epithelium in the characteristic aggregative pattern, forming a strong biofilm in a mucus layer, followed by cytotoxic and proinflammatory effects.260260 Vial PA, Robins-Browne R, Lior H, et al. Characterization of enteroadherent-aggregative Escherichia coli, a putative agent of diarrheal disease. J Infect Dis. 1988;158:70-79.,266266 Hicks S, Candy DC, Phillips AD. Adhesion of enteroaggregative Escherichia coli to pediatric intestinal mucosa in vitro. Infect Immun. 1996;64:4751-4760.

267 Nataro JP, Hicks S, Phillips AD, Vial PA, Sears CL. T84 cells in culture as a model for enteroaggregative Escherichia coli pathogenesis. Infect Immun. 1996;64:4761-4768.

268 Abe CM, Knutton S, Pedroso MZ, Freymüller E, Gomes TA. An enteroaggregative Escherichia coli strain of serotype O111:H12 damages and invades cultured T84 cells and human colonic mucosa. FEMS Microbiol Lett. 2001;203:199-205.

269 Andrade JA, Freymuller E, Fagundes-Neto U. Pathophysiology of enteroaggregative Escherichia coli infection: an experimental model utilizing transmission electron microscopy. Arq Gastroenterol. 2010;47:306-312.
-270270 Andrade JA, Freymuller E, Fagundes-Neto U. Adherence of enteroaggregative Escherichia coli to the ileal and colonic mucosa: an in vitro study utilizing the scanning electron microscopy. Arq Gastroenterol. 2011;48:199-204. Fragments from terminal ileum and colon excised from pediatric and adult patients were incubated with EAEC strains that were capable to colonize the ileal and colonic mucosa in the typical stacked-brick pattern over an augmented mucus layer.270270 Andrade JA, Freymuller E, Fagundes-Neto U. Adherence of enteroaggregative Escherichia coli to the ileal and colonic mucosa: an in vitro study utilizing the scanning electron microscopy. Arq Gastroenterol. 2011;48:199-204.

All these lines of evidence in combination with the identification of several putative virulence factors in prototype EAEC strains allowed the proposal of a three-stage model of EAEC pathogenesis: (a) abundant adherence to the intestinal mucosa, (b) production of cytotoxins and enterotoxins, and (c) induction of mucosal inflammation.211211 Navarro-Garcia F, Elias WP. Autotransporters and virulence of enteroaggregative E. coli. Gut Microbes. 2011;2:13-24. In the first stage, the contribution of fimbrial and afimbrial adhesins as well as other adhesive structures is essential. Several colonization factors have been identified in EAEC strains.271271 Torres AG, Zhou X, Kaper JB. Adherence of diarrheagenic Escherichia coli strains to epithelial cells. Infect Immun. 2005;73:18-29. In this stage, a characteristic increased secretion of mucus on the intestinal mucosa leads to the formation of a strong biofilm where EAEC are embedded.234234 Huang DB, Mohamed JA, Nataro JP, DuPont HL, Jiang ZD, Okhuysen PC. Virulence characteristics and the molecular epidemiology of enteroaggregative Escherichia coli isolates from travellers to developing countries. J Med Microbiol. 2007;56:1386-1392.,266266 Hicks S, Candy DC, Phillips AD. Adhesion of enteroaggregative Escherichia coli to pediatric intestinal mucosa in vitro. Infect Immun. 1996;64:4751-4760.,272272 Navarro-Garcia F, Gutierrez-Jimenez J, Garcia-Tovar C, Castro LA, Salazar-Gonzalez H, Cordova V. Pic, an autotransporter protein secreted by different pathogens in the Enterobacteriaceae family, is a potent mucus secretagogue. Infect Immun. 2010;78:4101-4109. In the following step, EAEC produce cytotoxic effects on the intestinal mucosa due to the secretion of toxins, inducing microvillus vesiculation, enlarged crypt openings, and increased epithelial cell extrusion.266266 Hicks S, Candy DC, Phillips AD. Adhesion of enteroaggregative Escherichia coli to pediatric intestinal mucosa in vitro. Infect Immun. 1996;64:4751-4760.,273273 Harrington SM, Dudley EG, Nataro JP. Pathogenesis of enteroaggregative Escherichia coli infection. FEMS MicrobioL Lett. 2006;254:12-18. EAEC-induced inflammation results from the strong colonization of the intestinal mucosa; however, all bacterial factors that contribute to this condition have not been identified. Inflammatory markers such as IL-8, IL-1β, interferon (INF)-γ and lactoferrin have been detected in stools of children and adults colonized by EAEC.274274 Steiner TS, Lima AA, Nataro JP, Guerrant RL. Enteroaggregative Escherichia coli produce intestinal inflammation and growth impairment and cause interleukin-8 release from intestinal epithelial cells. J Infect Dis. 1998;177:88-96.

275 Jiang ZD, Greenberg D, Nataro JP, Steffen R, DuPont HL. Rate of occurrence and pathogenic effect of enteroaggregative Escherichia coli virulence factors in international travelers. J Clin Microbiol. 2002;40:4185-4190.
-276276 Huang DB, DuPont HL, Jiang ZD, Carlin L, Okhuysen PC. Interleukin-8 response in an intestinal HCT-8 cell line infected with enteroaggregative and enterotoxigenic Escherichia coli. Clin Diagn Lab Immunol. 2004;11:548-551. Although this model summarizes the data so far obtained using in vivo, in vitro and ex vivo approaches it may not be valid for all strains.

A large foodborne outbreak of bloody diarrhea and HUS occurred in 2011 in Europe, affecting more than 4000 patients, most of them from Germany. This outbreak was caused by a Stx2-producing E. coli strain belonging to the serotype O104:H4. The genome of that strain was rapidly sequenced, revealing a unique hybrid combination of EAEC and STEC, i.e. the EAEC strain Ec55989 harboring the Shiga toxin 2-encoding prophage.166166 Bielaszewska M, Mellmann A, Zhang W, et al. Characterization of the E. coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study. Lancet Infect Dis. 2011;11:671-676.,213213 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. Several virulence factors of typical EAEC are present in that strain, including AggR, dispersin, Pic and ShET-1. Also expressed are two Shigella autotransporter proteins called SigA and SepA, implicated in mucosal damage and colonization.277277 Benjelloun-Touimi Z, Sansonetti PJ, Parsot C. SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion. Mol Microbiol. 1995;17:123-135.,278278 Al-Hasani K, Henderson IR, Sakellaris H, et al. The sigA gene which is borne on the she pathogenicity island of Shigella flexneri 2a encodes an exported cytopathic protease involved in intestinal fluid accumulation. Infect Immun. 2000;68:2457-2463. Interestingly, the EAEC Ec55989 is the prototype strain for AAF/III.237237 Bernier C, Gounon P, Le Bouguenec C. Identification of an aggregative adhesion fimbria (AAF) type III-encoding operon in enteroaggregative Escherichia coli as a sensitive probe for detecting the AAF-encoding operon family. Infect Immun. 2002;70:4302-4311. Conversely, the outbreak hybrid strain produces AAF/I, showing that the outbreak EAEC/STEC acquired an AAF/I-encoding plasmid.166166 Bielaszewska M, Mellmann A, Zhang W, et al. Characterization of the E. coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study. Lancet Infect Dis. 2011;11:671-676.,279279 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(7):e22751. It has been proposed that the presence of these virulence factors combined is responsible for the highly virulent attributes of that strain.166166 Bielaszewska M, Mellmann A, Zhang W, et al. Characterization of the E. coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study. Lancet Infect Dis. 2011;11:671-676.,213213 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.

Epidemiology

EAEC is an emerging pathogen affecting children and adults worldwide, responsible for cases of acute and persistent diarrhea. Nevertheless, the most important impact in terms of morbidity is among children younger than 5 years living in developing countries.214214 Hebbelstrup Jensen B, Olsen KE, Struve C, Krogfelt KA, Petersen AM. Epidemiology and clinical manifestations of enteroaggregative Escherichia coli. Clin Microbiol Rev. 2014;27:614-630. A meta-analysis study of the literature on the epidemiology of diarrhea that included the search of EAEC showed a statistical association of EAEC with acute and persistent diarrhea in developed and developing countries, with diarrhea in HIV-infected patients in developing countries, and adult traveler's diarrhea.280280 Huang DB, Nataro JP, DuPont HL, et al. Enteroaggregative Escherichia coli is a cause of acute diarrheal illness: a meta-analysis. Clin Infect Dis. 2006;43:556-563. In another meta-analysis study EAEC was associated with acute diarrhea in children living in South Asian countries.281281 Pabalan N, Singian E, Jarjanazi H, Steiner TS. Enteroaggregative Escherichia coli and acute diarrhea in children: a meta-analysis of South Asian populations. Eur J Clin Microbiol Infect Dis. 2013;32:597-607.

It is important to mention that data on the epidemiology of EAEC infection are somewhat inconsistent due to large variation in terms of method of detection, geographical location and patient age and socioeconomic status. Nonetheless, EAEC has been systematically identified as an emerging enteropathogen, strongly associated with acute and persistent diarrhea in children of developing countries. Moreover, in developed countries, EAEC have been frequently isolated from cases of diarrhea in children and adults in the last years.282282 Nataro JP, Mai V, Johnson J, et al. Diarrheagenic Escherichia coli infection in Baltimore, Maryland, and New Haven, Connecticut. Clin Infect Dis. 2006;43:402-407.,283283 Chattaway MA, Harris R, Jenkins C, et al. Investigating the link between the presence of enteroaggregative Escherichia coli and infectious intestinal disease in the United Kingdom, 1993 to 1996 and 2008 to 2009. Euro Surveill. 2013;18(37), pii: 20582.

In addition, several foodborne outbreaks of diarrhea caused by EAEC have been reported in Europe, Japan, Mexico and India.231231 Cobeljic M, Miljkovic-Selimovic B, Paunovic-Todosijevic D, et al. Enteroaggregative Escherichia coli associated with an outbreak of diarrhoea in a neonatal nursery ward. Epidemiol Infect. 1996;117:11-16.,232232 Itoh Y, Nagano I, Kunishima M, Ezaki T. Laboratory investigation of enteroaggregative Escherichia coli O untypeable:H10 associated with a massive outbreak of gastrointestinal illness. J Clin Microbiol. 1997;35:2546-2550.,284284 Eslava C, Villaseca J, Morales R, Navarro A, Cravioto A. Identification of a protein with toxigenic activity produced by enteroaggregative Escherichia coli. In: Abstracts of the 93rd General Meeting of the American Society for Microbiology. 1993.

285 Pai M, Kang G, Ramakrishna BS, Venkataraman A, Muliyil J. An epidemic of diarrhoea in south India caused by enteroaggregative Escherichia coli. Indian J Med Res. 1997;106:7-12.
-286286 Scavia G, Staffolani M, Fisichella S, et al. Enteroaggregative Escherichia coli associated with a foodborne outbreak of gastroenteritis. J Med Microbiol. 2008;57:1141-1146. One of them affected 2697 school children in Japan, after consumption of school lunches.232232 Itoh Y, Nagano I, Kunishima M, Ezaki T. Laboratory investigation of enteroaggregative Escherichia coli O untypeable:H10 associated with a massive outbreak of gastrointestinal illness. J Clin Microbiol. 1997;35:2546-2550.

Several studies have implicated EAEC as the predominant agent of persistent diarrhea in children.287287 Cravioto A, Tello A, Navarro A, et al. Association of Escherichia coli HEp-2 adherence patterns with type and duration of diarrhoea. Lancet. 1991;337:262-264.

288 Fang GD, Lima AA, Martins CV, Nataro JP, Guerrant RL. Etiology and epidemiology of persistent diarrhea in northeastern Brazil: a hospital-based, prospective, case-control study. J Pediatr Gastroenterol Nutr. 1995;21:137-144.
-289289 Wanke CA, Schorling JB, Barrett LJ, Desouza MA, Guerrant RL. Potential role of adherence traits of Escherichia coli in persistent diarrhea in an urban Brazilian slum. Pediatr Infect Dis J. 1991;10:746-751. EAEC-mediated persistent diarrhea has been linked to malnutrition and decrease in physical and intellectual development in several studies from Brazil.274274 Steiner TS, Lima AA, Nataro JP, Guerrant RL. Enteroaggregative Escherichia coli produce intestinal inflammation and growth impairment and cause interleukin-8 release from intestinal epithelial cells. J Infect Dis. 1998;177:88-96.,288288 Fang GD, Lima AA, Martins CV, Nataro JP, Guerrant RL. Etiology and epidemiology of persistent diarrhea in northeastern Brazil: a hospital-based, prospective, case-control study. J Pediatr Gastroenterol Nutr. 1995;21:137-144.,290290 Lima AA, Moore SR, Barboza MS, et al. Persistent diarrhea signals a critical period of increased diarrhea burdens and nutritional shortfalls: a prospective cohort study among children in northeastern Brazil. J Infect Dis. 2000;181:1643-1651. Notably, asymptomatic patients infected with EAEC also exhibit growth retardation.274274 Steiner TS, Lima AA, Nataro JP, Guerrant RL. Enteroaggregative Escherichia coli produce intestinal inflammation and growth impairment and cause interleukin-8 release from intestinal epithelial cells. J Infect Dis. 1998;177:88-96. Since its definition as a pathotype, high rates of asymptomatic young children carrying EAEC have been reported in several studies, involving subjects with low socioeconomic status in developing countries.214214 Hebbelstrup Jensen B, Olsen KE, Struve C, Krogfelt KA, Petersen AM. Epidemiology and clinical manifestations of enteroaggregative Escherichia coli. Clin Microbiol Rev. 2014;27:614-630. The persistence of EAEC may induce chronic intestinal inflammation, even in the absence of diarrhea, reducing its absorptive function and leading to malnutrition.274274 Steiner TS, Lima AA, Nataro JP, Guerrant RL. Enteroaggregative Escherichia coli produce intestinal inflammation and growth impairment and cause interleukin-8 release from intestinal epithelial cells. J Infect Dis. 1998;177:88-96.,291291 Opintan JA, Newman MJ, Ayeh-Kumi PF, et al. Pediatric diarrhea in southern Ghana: etiology and association with intestinal inflammation and malnutrition. Am J Trop Med Hyg. 2010;83:936-943. Growth impairment has also been observed in a mouse model of EAEC oral infection.292292 Roche JK, Cabel A, Sevilleja J, Nataro J, Guerrant RL. Enteroaggregative Escherichia coli (EAEC) impairs growth while malnutrition worsens EAEC infection: a novel murine model of the infection malnutrition cycle. J Infect Dis. 2010;202:506-514. Considering the high number of asymptomatic EAEC-colonized children in low-income countries, this pathotype has an important impact on public health as one cause of impaired physical and cognitive development.

EAEC is transmitted by the fecal-oral route by food or contaminated water.232232 Itoh Y, Nagano I, Kunishima M, Ezaki T. Laboratory investigation of enteroaggregative Escherichia coli O untypeable:H10 associated with a massive outbreak of gastrointestinal illness. J Clin Microbiol. 1997;35:2546-2550.,285285 Pai M, Kang G, Ramakrishna BS, Venkataraman A, Muliyil J. An epidemic of diarrhoea in south India caused by enteroaggregative Escherichia coli. Indian J Med Res. 1997;106:7-12.,286286 Scavia G, Staffolani M, Fisichella S, et al. Enteroaggregative Escherichia coli associated with a foodborne outbreak of gastroenteritis. J Med Microbiol. 2008;57:1141-1146. EAEC were detected in milk samples from infant feeding bottles that were handled by mothers with low socioeconomic status.293293 Morais TB, Gomes TAT, Sigulem DM. Enteroaggregative Escherichia coli in infant feeding bottles. Lancet. 1997;349:1448-1449. Also, viable EAEC were isolated in tabletop sauces from Mexican restaurants.294294 Adachi JA, Mathewson JJ, Jiang ZD, Ericsson CD, DuPont HL. Enteric pathogens in Mexican sauces of popular restaurants in Guadalajara, Mexico, and Houston, Texas. Ann Intern Med. 2002;136:884-887. No relationship has been found between EAEC strains isolated from humans and different animal species, indicating that animals may not represent a reservoir of human pathogenic typical EAEC.295295 Uber AP, Trabulsi LR, Irino K, et al. Enteroaggregative Escherichia coli from humans and animals differ in major phenotypical traits and virulence genes. FEMS Microbiol Lett. 2006;256:251-257.

EAEC has also emerged in the last years as an agent of urinary tract infections (UTI). Initially, Abe et al.,296296 Abe CM, Salvador FA, Falsetti IN, et al. Uropathogenic Escherichia coli (UPEC) strains may carry virulence properties of diarrhoeagenic E. coli. FEMS Immunol Med Microbiol. 2008;52:397-406. described the presence of EAEC virulence markers in strains isolated from UTI, which was subsequently observed by others.297297 Regua-Mangia AH, Irino K, da Silva Pacheco R, Pimentel Bezerra RM, Santos Périssé AR, Teixeira LM. Molecular characterization of uropathogenic and diarrheagenic Escherichia coli pathotypes. J Basic Microbiol. 2010;50(Suppl 1):S107-S115.

298 Park HK, Jung YJ, Chae HC, et al. Comparison of Escherichia coli uropathogenic genes (kps, usp and ireA) and enteroaggregative genes (aggR and aap) via multiplex polymerase chain reaction from suprapubic urine specimens of young children with fever. Scand J Urol Nephrol. 2009;43:51-57.

299 Nazemi A, Mirinargasi M, Merikhi N, Sharifi SH. Distribution of pathogenic genes aatA, aap, aggR, among uropathogenic Escherichia coli (UPEC) and their linkage with stbA gene. Indian J Microbiol. 2011;51:355-358.
-300300 Herzog K, Engeler Dusel J, Hugentobler M, et al. Diarrheagenic enteroaggregative Escherichia coli causing urinary tract infection and bacteremia leading to sepsis. Infect. 2014;42:441-444. Also, the presence of uropathogenic E. coli (UPEC) markers in EAEC collections has been reported.301301 Gomes TA, Abe CM, Marques LR. Detection of HeLa cell-detaching activity and alpha-hemolysin production in enteroaggregative Escherichia coli strains isolated from feces of Brazilian children. J Clin Microbiol. 1995;33:3364.,302302 Wallace-Gadsden F, Johnson JR, Wain J, Okeke IN. Enteroaggregative Escherichia coli related to uropathogenic clonal group A. Emerg Infect Dis. 2007;13:757-760. These findings pointed out the potential for some EAEC strains to cause UTI.

A community acquired UTI outbreak caused by an EAEC strain of serotype O78:H10, occurred in Denmark.303303 Olesen B, Scheutz F, Andersen RL, et al. Enteroaggregative Escherichia coli O78:H10, the cause of an outbreak of urinary tract infection. J Clin Microbiol. 2012;50:3703-3711. This multiresistant strain belonged to the multilocus sequence type ST10 and phylogenetic group A. This was the first time that EAEC was implicated as an agent of an outbreak of extraintestinal disease. The uropathogenic properties of this EAEC strain were conferred by specific virulence factors, such as the AAF/I fimbriae.304304 Boll EJ, Struve C, Boisen N, Olesen B, Stahlhut SG, Krogfelt KA. Role of enteroaggregative Escherichia coli virulence factors in uropathogenesis. Infect Immun. 2013;81:1164-1171. Recently, EAEC was implicated as a causative agent of one case of urosepsis.300300 Herzog K, Engeler Dusel J, Hugentobler M, et al. Diarrheagenic enteroaggregative Escherichia coli causing urinary tract infection and bacteremia leading to sepsis. Infect. 2014;42:441-444.

Detection and diagnosis

Among the DEC pathotypes, EAEC is the most difficult to categorize, since it is a very heterogeneous group. The defining characteristic of EAEC is the AA pattern in human epithelial cells or on a glass substrate in a distinctive stacked-brick formation. Thus, the gold standard method for distinguishing EAEC is to culture five E. coli colonies per patient in static Luria-broth at 37 °C, and then to infect semi-confluent HEp-2 cells for 3 or 6 h, looking for the typical AA pattern.212212 Nataro JP, Kaper JB, Robins-Browne R, Prado V, Vial P, Levine MM. Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells. Pediatr Infect Dis J. 1987;6:829-831.,305305 Rüttler ME, Yanzón CS, Cuitiño MJ, Renna NF, Pizarro MA, Ortiz AM. Evaluation of a multiplex PCR method to detect enteroaggregative Escherichia coli. Biocell. 2006;30:301-308. However, this test requires specialized facilities and is time-consuming, restricting its use only to research and certain reference laboratories.

Furthermore, despite that several protein components such as Pic, ShET1, EAST-1, and Pet are involved in the virulence of EAEC, none of them is present in all isolates. The presence of Pet in EAEC isolates was initially detected by immunoblotting assays after a preliminary step of culture supernatant concentration.256256 Navarro-Garcia F, Eslava C, Villaseca JM, et al. In vitro effects of a high-molecular-weight heat-labile enterotoxin from enteroaggregative Escherichia coli. Infect Immun. 1998;66:3149-3154. Vilhena-Costa et al.306306 Vilhena-Costa AB, Piazza RMF, Nara JM, Trabulsi LR, Martinez MB. Slot blot immunoassay as a tool for plasmid-encoded toxin detection in enteroaggregative Escherichia coli culture supernatants. Diagn Microbiol Infect Dis. 2006;55:101-106. developed a slot blot immunoassay that avoids the concentration step, allowing the detection of Pet directly from EAEC supernatant, after growing the EAEC bacterial isolate in TSB at 37 °C for 4 h. In this method, it was possible to evaluate Pet expression with specificity and reproducibility, using a rabbit polyclonal anti-Pet serum, which showed no cross-reaction with supernatants of non-Pet-expressing isolates and commensal E. coli.

Considering these difficulties, DNA probes were included as a valuable tool for EAEC detection.307307 Levine MM, Prado V, Robins-Browne R, et al. Use of DNA probes and HEp-2 cell adherence assay to detect diarrheagenic Escherichia coli. J Infect Dis. 1988;158:224-228. After sequencing the EcoRI-PstI fragment of pCVD432 (AA or EAEC probe) developed by Baudry et al.,228228 Baudry B, Savarino SJ, Vial P, Kaper JB, Levine MM. A sensitive and specific DNA probe to identify enteroaggregative Escherichia coli, a recently discovered diarrheal pathogen. J Infect Dis. 1990;161:1249-1251. primers complementary to this probe for PCR amplification were designed.308308 Schmidt H, Knop C, Franke S, Aleksic S, Heesemann J, Karch H. Development of PCR for screening of enteroaggregative Escherichia coli. J Clin Microbiol. 1995;33:701-705. This PCR assay was found to be a rapid, simple, and highly sensitive method, and therefore considered to be useful for screening stool specimens for the presence of EAEC strains. Rapid and practical multiplex PCR assays targeting more genes (aggR, aap and aatA, encoding the AggR regulator, dispersin and an ABC secretion system outer membrane protein, respectively) or aggR, pic and astA, encoding AggR, Pic and an EAST-1) have also been employed to detect EAEC strains.309309 Cerna JF, Nataro JP, Estrada-Garcia T. Multiplex PCR for detection of three plasmid-borne genes of enteroaggregative Escherichia coli strains. J Clin Microbiol. 2003;41:2138-2140.

310 Cordeiro F, Pereira DG, Rocha MR, Asensi MD, Elias WP, Campos LC. Evaluation of a multiplex PCR for identification of enteroaggregative Escherichia coli. J Clin Microbiol. 2008;46:828-829.
-311311 Antikainen J, Tarkka E, Haukka K, Siitonen A, Vaara M, Kirveskari J. New 16-plex PCR method for rapid detection of diarrheagenic Escherichia coli directly from stool samples. Eur J Clin Microbiol. 2009;28:899-908. Monteiro et al.248248 Monteiro BT, Campos LC, Sircili MP, et al. The dispersin-encoding gene (aap) is not restricted to enteroaggregative Escherichia coli. Diagn Microbiol Infect Dis. 2009;65:81-84. used PCR to evaluate aggR, aatA and aap in a collection of E. coli strains and found that aggR and aatA were more specific to EAEC than aap, suggesting that the simultaneous detection of aggR, aatA, and aaiA (a type VI secretion system protein) could be an improvement in the PCR detection of EAEC.

All these proposed PCR-based protocols detect plasmid genes, which disfavors the detection of atypical EAEC strains.305305 Rüttler ME, Yanzón CS, Cuitiño MJ, Renna NF, Pizarro MA, Ortiz AM. Evaluation of a multiplex PCR method to detect enteroaggregative Escherichia coli. Biocell. 2006;30:301-308.,309309 Cerna JF, Nataro JP, Estrada-Garcia T. Multiplex PCR for detection of three plasmid-borne genes of enteroaggregative Escherichia coli strains. J Clin Microbiol. 2003;41:2138-2140.,312312 Bouzari S, Jafari A, Zarepour M. Distribution of virulence related genes among enteroaggregative Escherichia coli isolates: using multiplex PCR and hybridization. Infect Genet Evol. 2005;5:79-83. Others, employing plasmid and chromosome loci, have not reported sensitivity and specificity of the assay.226226 Lima IF, Boisen N, Quetz Jda S, et al. Prevalence of enteroaggregative Escherichia coli and its virulence-related genes in a case-control study among children from north-eastern Brazil. J Med Microbiol. 2013;62:683-693.,313313 Jenkins C, Chart H, Willshaw GA, Cheasty T, Smith HR. Genotyping of enteroaggregative Escherichia coli and identification of target genes for the detection of both typical and atypical strains. Diagn Microbiol Infect Dis. 2006;55:13-19.,314314 Panchalingam S, Antonio M, Hossain A, et al. Diagnostic microbiologic methods in the GEMS-1 case/control study. Clin Infect Dis. 2012;55(Suppl 4):S294-S302. However, a multiplex PCR based on two genes encoded in the plasmid and two chromosome-borne genes is recommended to increase the ability to detect both typical and atypical EAEC strains. The aggR and aatA genes309309 Cerna JF, Nataro JP, Estrada-Garcia T. Multiplex PCR for detection of three plasmid-borne genes of enteroaggregative Escherichia coli strains. J Clin Microbiol. 2003;41:2138-2140.,313313 Jenkins C, Chart H, Willshaw GA, Cheasty T, Smith HR. Genotyping of enteroaggregative Escherichia coli and identification of target genes for the detection of both typical and atypical strains. Diagn Microbiol Infect Dis. 2006;55:13-19. and aaiA and aaiG genes315315 Dudley EG, Thomson NR, Parkhill J, Morin NP, Nataro JP. Proteomic and microarray characterization of the AggR regulon identifies a pheU pathogenicity island in enteroaggregative Escherichia coli. Mol Microbiol. 2006;61:1267-1282. incorporated in the assay detecting aaiA, aaiG, aggR and aatA demonstrated 94.8% sensitivity and 94.3% specificity, and the assay was able to effectively detect both groups of EAEC among E. coli isolated from stool cultures.316316 Andrade FB, Gomes TAT, Elias WP. A sensitive and specific molecular tool for detection of both typical and atypical enteroaggregative Escherichia coli. J Microbiol Methods. 2014;106:16-18. This method should improve EAEC detection, since this pathotype is responsible for acute and persistent diarrhea in children and adults and is also associated with foodborne diarrheal outbreaks.

Enterotoxigenic E. coli

ETEC strains are characterized by the production of colonization factors (CFs) and at least one of two enterotoxins: LT and ST. ETEC represents one of the most common causes of diarrhea in children in developing countries and in travelers to these regions. ETEC is also an economic burden to farmers and industry, where it is an important pathogen for broilers, swine, cattle and other farm animals. The group represents a highly diverse pathovar of diarrheiogenic E. coli, harboring mobile genetic elements such as plasmids and phages. ETEC heterogeneity was first demonstrated by phenotypic traits including the large diversity of lipopolysshacaride (LPS) and flagelin composition and the expression of different CFs and toxin types.317317 Gaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 1996;4:444-452.,318318 Wolf MK. Occurrence, distribution, and association of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli. Clin Microbiol Rev. 1997;10:569-584. Serological typing of ETEC strains have relied on the composition of outer membrane proteins and, mainly, in the somatic LPS (O) and flagellar (H) antigens.318318 Wolf MK. Occurrence, distribution, and association of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli. Clin Microbiol Rev. 1997;10:569-584.

319 Guth BE, Pacheco AB, von Krüger WM, Ferreira LCS. Comparison of outer membrane protein and lipopolysaccharide profiles of enterotoxigenic Escherichia coli strains isolated in São Paulo, Brazil. Braz J Med Biol Res. 1995;28:545-552.
-320320 Nishimura LS, Ferreira LCS, Pacheco ABF, Guth BE. Relationship between outer membrane protein and lipopolysaccharide profiles and serotypes of enterotoxigenic Escherichia coli isolated in Brazil. FEMS Microbiol Lett. 1996;143:253-258. ETEC comprise more than 100 somatic serogroups (O) and at least 34 flagellar types (H), combined in an unpredicted number of O:H serotypes, but only a limited number of serotypes are associated with infectious diseases, such as O8:H9, O6:H16, O78:H12 and O25:H42, and are therefore of major clinical relevance.318318 Wolf MK. Occurrence, distribution, and association of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli. Clin Microbiol Rev. 1997;10:569-584.,321321 Qadri F, Svennerholm AM, Faruque AS, Sack RB. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev. 2005;18:465-483.

The genetic diversity of ETEC has also been evaluated by molecular approaches including random amplification of polymorphic DNA (RAPD), MLEE, PFGE, multilocus sequence type (MLST) and whole-genome sequencing.322322 Pacheco ABF, Guth BEC, de Almeida DF, Ferreira LCS. Characterization of enterotoxigerfic Escherichia coli by random amplification of polymorphic DNA. Res Microbiol. 1996;147:175-182.

323 Pacheco ABF, Guth BEC, Soares KCC, de Almeida DF, Ferreira LCS. Clonal relationships among Escherichia coli serogroup O6 isolates based on RAPD. FEMS Microbiol Lett. 1997;148:255-260.

324 Pacheco ABF, Guth BEC, Soares KCC, Nishimura L, de Almeida DF, Ferreira LCS. Random amplification of polymorphic DNA reveals serotype-specific clonal clusters among enterotoxigenic Escherichia coli strains isolated from humans. J Clin Microbiol. 1997;35:1521-1525.

325 Pacheco ABF, Soares KCC, de Almeida DF, Viboud GI, Binsztein N, Ferreira LCS. Clonal nature of enterotoxigenic Escherichia coli serotype O6:H16 revealed by randomly amplified polymorphic DNA analysis. J Clin Microbiol. 1998;36:2099-2102.

326 Pacheco ABF, Ferreira LCS, Pichel MG, et al. Beyond serotypes and virulence-associated factors: detection of genetic diversity among O153:H45 CFA/I heat-stable enterotoxigenic Escherichia coli strains. J Clin Microbiol. 2001;39:4500-4505.

327 Regua-Mangia AH, Guth BC, Andrade JRC, Almeida DF, Binsztein N, Viboud GI. Genotypic and phenotypic characterization of enterotoxigenic Escherichia coli (ETEC) strains isolated in Rio de Janeiro city, Brazil. FEMS Immunol Med Microbiol. 2004;40:155-162.

328 Steinsland H, Lacher DW, Sommerfelt H, Whittam TS. Ancestral lineages of human enterotoxigenic Escherichia coli. J Clin Microbiol. 2010;48:2916-2924.

329 Sahl JW, Rasko DA. Analysis of global transcriptional profiles of enterotoxigenic Escherichia coli isolate E24377A. Infect Immun. 2012;80:1232-1242.
-330330 von Mentzer A, Connor TR, Wieler LH, et al. Identification of enterotoxigenic Escherichia coli (ETEC) clades with long-term global distribution. Nat Genet. 2014;46:1321-1326. More recently, 362 human-derived strains were subjected to next-generation whole-genome sequencing; 21 genotypes could be identified, and ETEC strains could be classified into 5 major phylogroups (A, B1, B2, D and E).330330 von Mentzer A, Connor TR, Wieler LH, et al. Identification of enterotoxigenic Escherichia coli (ETEC) clades with long-term global distribution. Nat Genet. 2014;46:1321-1326. Genetic analyses demonstrated that clonally related ETEC lineages sharing the same serotypes and CF and toxin profiles have worldwide distribution.327327 Regua-Mangia AH, Guth BC, Andrade JRC, Almeida DF, Binsztein N, Viboud GI. Genotypic and phenotypic characterization of enterotoxigenic Escherichia coli (ETEC) strains isolated in Rio de Janeiro city, Brazil. FEMS Immunol Med Microbiol. 2004;40:155-162.,328328 Steinsland H, Lacher DW, Sommerfelt H, Whittam TS. Ancestral lineages of human enterotoxigenic Escherichia coli. J Clin Microbiol. 2010;48:2916-2924.,330330 von Mentzer A, Connor TR, Wieler LH, et al. Identification of enterotoxigenic Escherichia coli (ETEC) clades with long-term global distribution. Nat Genet. 2014;46:1321-1326.

331 Isidean SD, Riddle MS, Savarino SJ, Porter CK. A systematic review of ETEC epidemiology focusing on colonization factor and toxin expression. Vaccine. 2011;29:6167-6178.
-332332 Joffré E, Sjöling A. The LT1 and LT2 variants of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) are associated with major ETEC lineages. Gut Microbes. 2016;7:75-81. On the other hand, genetically distinct ETEC strains, frequently found among asymptomatic subjects show high antigen heterogeneity with regard to virulence traits and serotypes.333333 Lasaro MA, Rodrigues JF, Mathias-Santos C, et al. Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans. J Bacteriol. 2008;190:2400-2410. Apparently, these strains have recently acquired the genes encoding virulence-associated traits, and their maintenance is driven by selective pressure.328328 Steinsland H, Lacher DW, Sommerfelt H, Whittam TS. Ancestral lineages of human enterotoxigenic Escherichia coli. J Clin Microbiol. 2010;48:2916-2924.,330330 von Mentzer A, Connor TR, Wieler LH, et al. Identification of enterotoxigenic Escherichia coli (ETEC) clades with long-term global distribution. Nat Genet. 2014;46:1321-1326.

Virulence factors, mechanisms and pathogenesis

Following the initial discovery of the association of ETEC with diarrheic disease in humans in the 1950s, there was an intense effort to identify ETEC virulence-associated traits that could help to understand the physiology of the pathological process and lead to the development of specific diagnostic methods. ETEC strains characteristically produce adhesins, or CFs, proteinaceus complex that may take the shape of fimbrial, fibrillar or nonfimbrial structures on the bacterial surface. The adhesins expressed by ETEC strains facilitate the adherence of the bacteria to the intestinal mucosa and confer host specificity to the different strains.317317 Gaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 1996;4:444-452.,321321 Qadri F, Svennerholm AM, Faruque AS, Sack RB. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev. 2005;18:465-483.

Approximately 30 antigenically distinct CFs have been identified in clinically relevant ETEC strains, but only a few are usually found among samples collected from diarrheic patients.317317 Gaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 1996;4:444-452.,330330 von Mentzer A, Connor TR, Wieler LH, et al. Identification of enterotoxigenic Escherichia coli (ETEC) clades with long-term global distribution. Nat Genet. 2014;46:1321-1326. Besides differences regarding biogenesis and structural organization, ETEC CFs show specific antigenic, genetic and biochemical features, which are currently used to cluster them into three main groups: the colonization factor antigen I (CFA/I)-like group, the coli surface antigen 5 (CS5)-like group and the class 1b group.317317 Gaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 1996;4:444-452.,334334 Nada RA, Shaheen HI, Khalil SB, et al. Discovery and phylogenetic analysis of novel members of class b enterotoxigenic Escherichia coli adhesive fimbriae. J Clin Microbiol. 2011;49:1403-1410.,335335 von Mentzer A, Thesis Whole Genome Sequencing of Enterotoxigenic Escherichia coli (ETEC): Identification of ETEC Lineages and Novel Colonization Factors. Printed in Gothenburg, Sweden: Department of Microbiology and Immunology, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg; 2016. The CFA/I-like group harbors the first described CF (CFA/I) and some of the most clinically prevalent CFs, including CS1, CS2, CS4, CS14, CS17, CS19 and putative colonization factor O71(PCFO71), while the CS5-like group comprises only CS5 and CS7. The class 1b group includes CS12, CS18, CS20 and the recently described CS26-28 and CS30 types.317317 Gaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 1996;4:444-452.,334334 Nada RA, Shaheen HI, Khalil SB, et al. Discovery and phylogenetic analysis of novel members of class b enterotoxigenic Escherichia coli adhesive fimbriae. J Clin Microbiol. 2011;49:1403-1410.,335335 von Mentzer A, Thesis Whole Genome Sequencing of Enterotoxigenic Escherichia coli (ETEC): Identification of ETEC Lineages and Novel Colonization Factors. Printed in Gothenburg, Sweden: Department of Microbiology and Immunology, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg; 2016. Additionally, genetic relationships are also observed between strains expressing CS8 and CS21, CS13 and CS23, as well as between strains expressing CS15 and CS22.336336 Taniguchi T, Fujino Y, Yamamoto K, Honda T. Sequencing of the gene encoding the major pilin of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli and evidence that CFA/III is related to type IV pili. Infect Immun. 1995;63:724-728.

337 Pichel M, Binsztein N, Viboud G. CS22, a novel human enterotoxigenic Escherichia coli adhesin, is related to CS15. Infect Immun. 2000;68:3280-3285.
-338338 Del Canto F, Botkin DJ, Valenzuela P, et al. Identification of coli surface antigen 23, a novel adhesin of enterotoxigenic Escherichia coli. Infect Immun. 2012;80:2791-2801. Other previously characterized CFs, such as CS3, CS6, CS10 and CS11, are not classified into the known CS families.317317 Gaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 1996;4:444-452. Some CFs, such as CS18 and CS20, are related to swine-derived ETEC fimbriae, which show a lower heterogeneity than those found in strains isolated from humans.317317 Gaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 1996;4:444-452.,321321 Qadri F, Svennerholm AM, Faruque AS, Sack RB. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev. 2005;18:465-483.,339339 Francis DH. Enterotoxigenic Escherichia coli infection in pigs and its diagnosis. J Swine Health Prod. 2002;10:171-175. Strains expressing CFA/I, CFA-II (CS1/CS3, CS2/CS3 or CS3), CFA-IV (CS4/CS6, CS5/CS6 or CS6), CS17 and/or CS21 are the most prevalent CFs found in epidemiological studies, whereas other CFs are found in ETEC strains not clearly linked to diarrheal disease.317317 Gaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 1996;4:444-452.,318318 Wolf MK. Occurrence, distribution, and association of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli. Clin Microbiol Rev. 1997;10:569-584.,331331 Isidean SD, Riddle MS, Savarino SJ, Porter CK. A systematic review of ETEC epidemiology focusing on colonization factor and toxin expression. Vaccine. 2011;29:6167-6178.

After adherence to the intestinal mucosa, ETEC strains produce enterotoxins, which are recognized as the second component associated with diarrheal disease. Two major categories of enterotoxins have been identified among ETEC strains, isolated either from humans or other animal hosts: LT and ST. Both toxin types mediate deregulation of membrane ion channels in the epithelial membrane, leading to the loss of ions and massive amounts of water, the major characteristic of watery diarrhea caused by these bacterial strains.340340 Fleckenstein JM, Hardwidge PR, Munson GP, Rasko DA, Sommerfelt H, Steinsland H. Molecular mechanisms of enterotoxigenic Escherichia coli infection. Microbes Infect. 2010;12:89-98.

LT are composed of five identical monomers (11.5 kDa) arranged in a ring shape to form a pentameric B subunit, and a 28-kDa A subunit linked to the B subunit by the helical A2 domain. The B subunit binds to cell surface receptors, particularly to gangliosides, promoting toxin internalization and retrograte transport up to the endoplasmic reticulum, where the A1 domain is cleaved from the A2 domain and released to the cytoplasm. The A1 domain transfers the ADP-ribose moiety from the NAD+ cofactor to stimulatory G protein, which becomes active and capable of stimulating adenylate cyclase, leading to an intracellular increase in cyclic adenosine monophosphate (cAMP). Higher cAMP levels in the cell induce protein kinase A activation, which in turn leads to phosphorylation of ion channels, resulting in Cl release as well as decrease in Na+ uptake and, consequently, massive water release to intestinal lumen, the major characteristic of secretory diarrhea caused by these pathogens.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201.,341341 Spangler BD. Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol Rev. 1992;56:622-647. ST, a monomeric protein of about 5 kDa, may also induce osmotic deregulation, activating directly the guanylate cyclase C located at the apical membrane of the intestinal cells to produce intracellular cyclic guanosine monophosphate and consequently to generate secretion of Cl ions and water from the intestinal epithelium. However, an ST variant first isolated from pigs shows distinct physiological activity, characterized by the loss of villus epithelial cells and net bicarbonate secretion.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201. The toxins LT and ST, separately or in combination, are able to induce cellular water-electrolyte imbalance, which surely contributes to ETEC pathogenesis.

A hallmark in ETEC biology is the expression of enterotoxins, which also display a significant antigenic heterogeneity. Approximately one-third of the strains isolated from diarrheic patients express only LT or only ST, while another third express both toxin types. In addition, two unrelated ST groups, with different functional and structural features, have been identified: (i) STa, comprising two variants (STh and STp) associated with human disease, and (ii) STb, which is generally found among swine-derived ETEC strains. Similarly, LT are divided into two antigenically distinct groups: LT-I and LT-II.66 Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11(1):142-201. Initially, two LT-I variants, isolated from human or swine-derived ETECs (LTh and LTp, respectively), were described and shown to have high amino acid sequence identity and similar but not equal antigenicity and biochemical and receptor-binding properties.342342 Yamamoto T, Yokota T. Sequence of heat-labile enterotoxin of Escherichia coli pathogenic for humans. J Bacteriol. 1983;155:728-733.,343343 Grange PA, Parrish LA, Erickson AK. Expression of putative Escherichia coli heat-labile enterotoxin (LT) receptors on intestinal brush borders from pigs of different ages. Vet Res Commun. 2006;30:57-71. The related LT-II variants (LT-IIa,-IIb,-IIc) have been isolated from human beings or other hosts and contaminated food and bind to different receptors.344344 Guth BE, Pickett CL, Twiddy EM, et al. Production of type II heat-labile enterotoxin by Escherichia coli isolated from food and human feces. Infect Immun. 1986;54:587-589.

345 Guth BE, Twiddy EM, Trabulsi LR, Holmes RK. Variation in chemical properties and antigenic determinants among type II heat-labile enterotoxins of Escherichia coli. Infect Immun. 1986;54:529-536.

346 Hajishengallis G, Arce S, Gockel CM, Connell TD, Russell MW. Immunomodulation with enterotoxins for the generation of secretory immunity or tolerance: applications for oral infections. Crit Rev Oral Biol Med. 2005;84:1104-1116.
-347347 Nawar HF, King-Lyons ND, Hu JC, Pasek RC, Connell TD. LT-IIc, a new member of the type II heat-labile enterotoxin family encoded by an Escherichia coli strain obtained from a non-mammalian host. Infect Immun. 2010;78:4705-4713. The LT-IIa, LT-IIb and LT-IIc share 51, 52 and 49% or 15, 16 and 7% identity with LT-Ih regarding the A and B subunits, respectively.347347 Nawar HF, King-Lyons ND, Hu JC, Pasek RC, Connell TD. LT-IIc, a new member of the type II heat-labile enterotoxin family encoded by an Escherichia coli strain obtained from a non-mammalian host. Infect Immun. 2010;78:4705-4713.,348348 Jobling MG, Holmes RK. Type II heat-labile enterotoxins from 50 diverse Escherichia coli isolates belong almost exclusively to the LT-IIc family and may be prophage encoded. PLoS ONE. 2012;7:e29898.

More recently, a pioneer study carried out with ETEC strains isolated in Brazil demonstrated a rather high intraspecific LTh variability between LT-producing ETEC strains.333333 Lasaro MA, Rodrigues JF, Mathias-Santos C, et al. Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans. J Bacteriol. 2008;190:2400-2410.,349349 Lasaro MA, Mathias-Santos C, Rodrigues JF, Ferreira LCS. Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC). FEMS Immunol Med Microbiol. 2009;55:93-99.,350350 Rodrigues JF, Mathias-Santos C, Sbrogio-Almeida ME, et al. Functional diversity of heat-labile toxins (LT) produced by enterotoxigenic Escherichia coli: differential enzymatic and immunological activities of LT1 (hLT) and LT4 (pLT). J Biol Chem. 2011;286:5222-5233. In a collection of 51 ETEC strains expressing LT and/or ST, 50 genetic polymorphic sites were found in the LT-encoding genes, which revealed 16 natural LT variants according to differences in amino acid sequences. Among these variants, named LT1 to LT16, two (LT1 and LT2) were associated with a limited number of serotypes with a global distribution and mainly isolated from diarrheic patients.333333 Lasaro MA, Rodrigues JF, Mathias-Santos C, et al. Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans. J Bacteriol. 2008;190:2400-2410. In contrast, most of the detected LT variants were observed among LT-producing ETEC strains isolated from asymptomatic subjects.333333 Lasaro MA, Rodrigues JF, Mathias-Santos C, et al. Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans. J Bacteriol. 2008;190:2400-2410. More recently, 12 additional LT types were identified in a larger collection of ETEC strains isolated from different regions of the world.351351 Joffre E, von Mentzer A, Abd El Ghany M, et al. Allele variants of enterotoxigenic Escherichia coli heat-labile toxin are globally transmitted and associated with colonization factors. J Bacteriol. 2015;197:392-403. Interestingly, a much reduced genetic variability was found in the LT-encoding genes among ETEC strains isolated from pigs (LTp) and ST-encoding genes.352352 Zhang C, Rausch D, Zhang W. Little heterogeneity among genes encoding heat-labile and heat-stable toxins of enterotoxigenic Escherichia coli strains isolated from diarrheal pigs. Appl Environ Microbiol. 2009;75:6402-6405.,353353 Joffré E, von Mentzer A, Svennerholm AM, Sjöling Å. Identification of new heat-stable (STa) enterotoxin allele variants produced by human enterotoxigenic Escherichia coli (ETEC). Int J Med Microbiol. 2016;306:586-594.

The natural diversity of LT types found among ETEC strains isolated from symptomatic and asymptomatic humans suggests that some LT types can show higher toxicity to eukaryotic cells and can be expressed at different levels compared to other toxin types. Indeed, previous observations indicated that some LT types are endowed with different toxicity, under in vitro and in vivo conditions.333333 Lasaro MA, Rodrigues JF, Mathias-Santos C, et al. Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans. J Bacteriol. 2008;190:2400-2410.,349349 Lasaro MA, Mathias-Santos C, Rodrigues JF, Ferreira LCS. Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC). FEMS Immunol Med Microbiol. 2009;55:93-99.,350350 Rodrigues JF, Mathias-Santos C, Sbrogio-Almeida ME, et al. Functional diversity of heat-labile toxins (LT) produced by enterotoxigenic Escherichia coli: differential enzymatic and immunological activities of LT1 (hLT) and LT4 (pLT). J Biol Chem. 2011;286:5222-5233. A natural LT variant, similar to the LT expressed by swine-derived strains, showed reduced toxicity due to an amino acid replacement at a key polymorphic site in the A subunit.333333 Lasaro MA, Rodrigues JF, Mathias-Santos C, et al. Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans. J Bacteriol. 2008;190:2400-2410.,350350 Rodrigues JF, Mathias-Santos C, Sbrogio-Almeida ME, et al. Functional diversity of heat-labile toxins (LT) produced by enterotoxigenic Escherichia coli: differential enzymatic and immunological activities of LT1 (hLT) and LT4 (pLT). J Biol Chem. 2011;286:5222-5233. This amino acid change provided a less flexible A subunit structure, impairing appropriate contact with the cofactor (NAD+) at the catalytic site.350350 Rodrigues JF, Mathias-Santos C, Sbrogio-Almeida ME, et al. Functional diversity of heat-labile toxins (LT) produced by enterotoxigenic Escherichia coli: differential enzymatic and immunological activities of LT1 (hLT) and LT4 (pLT). J Biol Chem. 2011;286:5222-5233. Other authors observed that natural polymorphisms in the B subunit resulted in decreased receptor binding and therefore reduced toxicity to eukaryotic cells.352352 Zhang C, Rausch D, Zhang W. Little heterogeneity among genes encoding heat-labile and heat-stable toxins of enterotoxigenic Escherichia coli strains isolated from diarrheal pigs. Appl Environ Microbiol. 2009;75:6402-6405. These results suggest that the presence of ETEC strains expressing different LT variants may correlate with the incidence of symptoms among infected subjects, particularly among infected infants not previously exposed to ETEC infections.

Variable LT expression may also impact the severity of ETEC-associated disease. Previous observations demonstrated that the amounts of LT produced and/or secreted by ETEC are dramatically different among strains and clinical isolates.332332 Joffré E, Sjöling A. The LT1 and LT2 variants of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) are associated with major ETEC lineages. Gut Microbes. 2016;7:75-81.,351351 Joffre E, von Mentzer A, Abd El Ghany M, et al. Allele variants of enterotoxigenic Escherichia coli heat-labile toxin are globally transmitted and associated with colonization factors. J Bacteriol. 2015;197:392-403.,354354 Lasaro MAS, Rodrigues JF, Mathias-Santos C, et al. Production and release of heat-labile toxin by wild-type human-derived enterotoxigenic Escherichia coli. FEMS Immunol Med Microbiol. 2006;48:123-131.

355 Lasaro MAS, Rodrigues JF, Cabrera-Crespo J, Sbrogio-Almeida ME, Lasaro MO, Ferreira LCS. Evaluation of experimental conditions for quantification of lt produced by human derived enterotoxigenic Escherichia coli strains. Braz J Microbiol. 2007;38:446-451.
-356356 Rocha LB, Ozaki CY, Horton DS, et al. Different assay conditions for detecting the production and release of heat-labile and heat-stable toxins in enterotoxigenic Escherichia coli isolates. Toxins. 2013;5:2384-2402. The presence of single nucleotide changes in the etx operon regulatory region may be found and, at least for some of them, are associated with different transcriptional and translational activity among wild ETEC strains.332332 Joffré E, Sjöling A. The LT1 and LT2 variants of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) are associated with major ETEC lineages. Gut Microbes. 2016;7:75-81.,unpublished data Nonetheless, further studies are required to demonstrate a clear link between transcriptional and post-transcriptional events and the severity of the symptoms associated with ETEC infection.

Epidemiology

Annually, infections with different ETEC strains cause an astonishing number of diarrheal episodes, greatly exceeding 200 million cases and causing approximately 75,000 deaths, mainly among babies and young children in tropical areas with poor sanitary conditions.118118 Kotloff KL, Nataro JP, Blackwelder W, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicentre Study, GEMS): a prospective, case-control study. Lancet. 2013;382(9888):209-222.,357357 Lamberti LM, Bourgeois AL, Walker CLF, Black RE, Sack D. Estimating diarrheal illness and deaths attributable to Shigellae and enterotoxigenic Escherichia coli among older children, adolescents, and adults in South Asia and Africa. PLoS Negl Trop Dis. 2014;8:e2705. In Brazil, epidemiological data harvested at different times between 1978 and 2007 have demonstrated that the incidence of ETEC-induced diarrhea ranges from 3.5 to 20.45%.115115 Franzolin MR, Alves RCB, Keller R, et al. Prevalence of diarrheagenic Escherichia coli in children with diarrhea in Salvador, Bahia, Brazil. Mem Inst Oswaldo Cruz. 2005;100(4):359-363.,358358 Reis MHL, Castro AFP, Toledo MRF, Trabulsi LR. Production of heat-stable enterotoxin by the 0128 serogroup of Escherichia coli. Infect Immun. 1979;24:289-290.

359 Gatti MSV, Ricci LC, Serafim MB, De Castro AF. Incidência de Escherichia coli enterotoxigênica (ETEC), rotavirus e Clostridium perfringens de casos de diarreia em crianças, na região de Campinas, SP, Brasil. Rev Inst Med Trop S Paulo. 1989;31:392-398.

360 Gomes TAT, Rassi V, Mac Donald KL, et al. Enteropathogens associated with acute diarrheal disease in urban infants in São Paulo, Brazil. J Infect Dis. 1991;164:331-337.
-361361 Regua-Mangia AH, Gomes TAT, Vieira MAM, Andrade JR, Irino K, Teixeira LM. Frequency and characteristics of diarrhoeagenic Escherichia coli strains isolated from children with and without diarrhoea in Rio de Janeiro, Brazil. J Infect. 2004;48:161-167.

Detection and diagnosis

This pathotype is mainly characterized by the enterotoxins it produces, and diagnosis depends upon identifying either LT and/or ST. One or both toxins may be expressed by ETEC strains.340340 Fleckenstein JM, Hardwidge PR, Munson GP, Rasko DA, Sommerfelt H, Steinsland H. Molecular mechanisms of enterotoxigenic Escherichia coli infection. Microbes Infect. 2010;12:89-98.,362362 Reis MHL, Guth BEC, Gomes TAT, Murahovschi J, Trabulsi LR. Frequency of Escherichia coli strains producing heat-labile toxin or heat-stable toxin or both in children with and without diarrhea in Sao Paulo. J Clin Microbiol. 1982;15:1062-1064.

363 Torres ME, Pírez MC, Schelotto F, et al. Etiology of children's diarrhea in Montevideo, Uruguay: associated pathogens and unusual isolates. J Clin Microbiol. 2001;39:2134-2139.
-364364 Sack RB. The discovery of cholera - like enterotoxins produced by Escherichia coli causing secretory diarrhoea in humans. Indian. J. Med. Res. 2011;133:171-178. The diagnosis of ETEC strains should include, in addition to LT and ST detection, complementary PCR assays for the detection of virulence genes such as clyA, eatA, tia, tibC, leoA, and east-1.340340 Fleckenstein JM, Hardwidge PR, Munson GP, Rasko DA, Sommerfelt H, Steinsland H. Molecular mechanisms of enterotoxigenic Escherichia coli infection. Microbes Infect. 2010;12:89-98. A sensitive and specific PCR assay with primers targeting the genes lt and st was reported by Stacy-Phipps et al.,365365 Stacy-Phipps S, Mecca JJ, Weiss JB. Multiplex PCR assay and simple preparation method for stool specimes detect enterotoxigenic Escherichia coli DNA during course of infection. J Clin Microbiol. 1995;33:1054-1059. and later by Youmans et al.,366366 Youmans BP, Ajami NJ, Jiang ZD, Petrosino JF, DuPont HL, Highlander SK. Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers’ diarrhea samples. Am J Trop Med Hyg. 2014;90:124-132. using quantitative real-time PCR. Moreover, several multiplex PCR assays were also developed using these two genes.367367 Toma C, Lu Y, Higa N, et al. Multiplex PCR assay for identification of human diarrheagenic Escherichia coli. J Clin Microbiol. 2003;41:2669-2671.

368 Vidal R, Vidal M, Lagos R, Levine M, Prado V. Multiplex PCR for diagnosis of enteric infetions associated with diarrheagenic Escherichia coli. J Clin Microbiol. 2004;42:1787-1789.
-369369 Aranda KR, Fabbricott SH, Fagundes-Neto U, Scaletsky IC. Single multiplex assay to identify simultaneously enteropathogenic, enteroaggregative, enterotoxigenic, enteroinvasive, and Shiga toxin-producing Escherichia coli strains in Brazilian children. FEMS Microbiol Lett. 2007;267:145-150.

Phenotypical detection of ETEC was initially performed using supernatants obtained from single E. coli colonies and by laborious procedures such as rabbit ileal loop test,370370 Evans DG, Evans DJ, Pierce NF. Differences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of Escherichia coli. Infect Immun. 1973;7:873-880. suckling mouse assay371371 Dean AG, Ching YC, Williams RG, Harden LB. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J Infect Dis. 1972;125:407-411. or cytopathic effect studies on CHO or Y1 adrenal cell monolayers, in which the presence of LT in supernatants was indicated by rounding of Y1 cells or elongation of CHO cells after 24 h of incubation.372372 Donta ST, Moon HW, Whipp SC. Detection of heat-labile Escherichia coli enterotoxin with the use of adrenal cells in tissue culture. Science. 1974;183:334-336.,373373 Guerrant RL, Brunton LL, Schnaitman TC, Rebhun LI, Gilman AG. Cyclic adenosine monophosphate and alteration of Chinese hamster ovary cell morphology: a rapid, sensitive in vitro assay for the enterotoxins of Vibrio cholerae and Escherichia coli. Infect Immun. 1974;10:320-327.

A number of immunoassays have been developed for ST detection, including radioimmunoassay and enzyme-linked immunosorbent assay (ELISA). Both tests correlate well with results obtained with the suckling-mouse assay and require substantially less expertise.374374 Giannella RA, Drake KW, Luttrell M. Development of a radioimmunoassay for Escherichia coli heat-stable enterotoxin. Infect Immun. 1981;33:186-192.,375375 Cryan B. Comparison of three assay systems for detection of enterotoxigenic Escherichia coli heat-stable enterotoxin. J Clin Microbiol. 1990;28:792-794. ELISA assays were then developed using the GM1 receptor to bind LT obtained from filtered culture supernatants or employing a competitive test for LT, which replaced former procedures.376376 Bläck E, Svennerholm AM, Holmgren J, Möllby R. Evaluation of a ganglioside immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin. J Clin Microbiol. 1979;10:791-795.

Immunological assays for LT detection includes the traditional Biken test, latex agglutination, and reliable and easy to perform commercially available tests, such as the reversed passive latex agglutination and the staphylococcal coagglutination test.321321 Qadri F, Svennerholm AM, Faruque AS, Sack RB. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev. 2005;18:465-483. Several immunological assays where LT is captured either by ganglioside GM1 (its receptor in the host cell) or by antibodies have been described.139139 Piazza RMF, Abe CM, Horton DSPQ, et al. Detection and subtyping methods of diarrheagenic Escherichia coli strains. In: Torres AG, ed. Pathogenic Escherichia coli in Latin America. Bentham Science Publishers; 2010:95-115.,321321 Qadri F, Svennerholm AM, Faruque AS, Sack RB. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev. 2005;18:465-483.,377377 Menezes CA, Imamura SY, Trabulsi LR, et al. Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli. Mem Inst Oswaldo Cruz. 2006;101:875-880.,378378 Menezes CA, Gonçalves DS, Amianti J, et al. Capture immunoassay for LT detection produced by enterotoxigenic Escherichia coli in bacterial isolates. Braz J Microbiol. 2003;34:11-13. Assays for ST by indirect ELISA using IgG1 ST-mAb and for LT by capture ELISA employing IgG enriched fraction of a rabbit polyclonal as a capture antibody and IgG2b LT-mAb as a second antibody have been employed as tools for diagnosis. The presence of bile salts and the use of certain antibiotics improved ETEC toxin production/release. Triton X-100, as chemical treatment, proved to be an alternative method for toxin release. Consequently, a common protocol that can increase the production and release of LT and ST could facilitate and enhance the sensitivity of diagnostic tests for ETEC.355355 Lasaro MAS, Rodrigues JF, Cabrera-Crespo J, Sbrogio-Almeida ME, Lasaro MO, Ferreira LCS. Evaluation of experimental conditions for quantification of lt produced by human derived enterotoxigenic Escherichia coli strains. Braz J Microbiol. 2007;38:446-451. Afterwards, those monoclonal antibodies were rebuilt resulting in single chain fragment variable (scFv) fragments. The developed recombinant scFvs against LT and ST constitute a promising starting point for simple and cost-effective ETEC diagnosis.379379 Ozaki CY, Silveira CRF, Andrade FB, et al. Single chain variable fragments produced in Escherichia coli against heat-labile and heat-stable toxins from enterotoxigenic E. coli. PLOS ONE. 2015;10:e0131484.

Enteroinvasive E. coli

Enteroinvasive E. coli (EIEC) is a causative agent of dysentery in humans, especially in developing countries.380380 Trabulsi LR, Toledo MRF, Murahovschi J, Fagundes Neto U, Candeias JAM. Epidemiology of diarrhoea disease in South America. In: Tzipori S, ed. Infectious Diarrhoea in the Young. Amsterdam: Elsevier Biol. Med. Press; 1985:121-125. It causes keratoconjunctivitis in experimental guinea pigs381381 Silva RM, Toledo MRF, Trabulsi LR. Biochemical and cultural characteristics of invasive Escherichia coli. J Clin Microbiol. 1980;11:441-444. and invades human colon cells, causing an infection similar to that caused by Shigella sp.382382 Formal SB, Hornik RB. Invasive Escherichia coli. J Infect Dis. 1978;137:641-644.,383383 Formal SB, Hale TL, Sansonetti PJ. Invasive enteric pathogens. Rev Infect Dis. 1983;5:702-707. The first description of EIEC was performed by EWING and GRAWATTI in 1947.384384 Ewing WH, Gravatti JL. Shigella types encountered in the mediterranean area. J Bacteriol. 1947;53:191-195. The first works emphasizing the particular biochemical characteristics of EIEC samples were presented in 1967 by Trabulsi et al.,385385 Trabulsi LR, Fernandes MRF. Antigenic identity of culture 193T-64 and E.coli O136:K78 (B22). Rev Inst Med Trop. 1969;11:101-103. in Brazil and by Sakazaki et al.,386386 Sakazaki R, Tamura K, Saito M. Enteropathogenic Escherichia coli associated with diarrhea in children and adults. Jpn J Med Sci Biol. 1967;20:387-399. in Japan. All isolates studied were Serény test positive (guinea pig keratoconjunctivitis) and the strains were lysine decarboxylase negative, late fermenting lactose and generally non-motile, except for samples of the O124 serogroup. The study of the biochemical behavior of 97 samples of EIEC381381 Silva RM, Toledo MRF, Trabulsi LR. Biochemical and cultural characteristics of invasive Escherichia coli. J Clin Microbiol. 1980;11:441-444. corroborated the results obtained previously. It has been shown that this group of diarrheagenic E. coli belonged to well-defined bioserotypes, O28ac:H-, O29:H-, O112ac:H-, O121:H-, O124:H-, O124:H30, O135:H-, O136:H-, O143:H, O144:H-, O152:H-, O159:H-, O164:H-, O167:H- and O173:H-.381381 Silva RM, Toledo MRF, Trabulsi LR. Biochemical and cultural characteristics of invasive Escherichia coli. J Clin Microbiol. 1980;11:441-444.,387387 Orskov I, Wachsmuth K, Taylor DN. Two new Escherichia coli O groups: O172 from ‘shiga-like’ toxin II-producing strains (EHEC) and O173 from enteroinvasive E. coli (EIEC). APMIS. 1991;99:30-32.

388 Matsushita S, Yamada S, Kai A, Kudoh Y. Invasive strains of Escherichia coli belonging to serotype O121:NM. J Clin Microbiol. 1993;31(11):3034-3035.
-389389 Vöros S, Rédey B, Csizmazia F. Antigenic structure of a new enteropathogenic Escherichia coli strains. Acta Microbiol Acad Sci Hung. 1964;11:125-129. In 1964, it was demonstrated that samples of the O32 and O42 serogroups of E. coli also had the ability to cause keratoconjunctivitis in guinea pigs.389389 Vöros S, Rédey B, Csizmazia F. Antigenic structure of a new enteropathogenic Escherichia coli strains. Acta Microbiol Acad Sci Hung. 1964;11:125-129. However, the existence of enteroinvasive bioserotypes in O42 serogroup was not confirmed, and O32 bioserotype is actually an aerogenic variant of S. boydii 14, as shown by Toledo et al.390390 Toledo MRF, Silva RM, Trabulsi LR. Sachs “Enterobacterium A12” is an aerogenic variant of Shigella boydii 14. Int J Syst Bacteriol. 1981;31:242-244. There are reports of isolation of EIEC samples belonging to other mobile serotypes, O144H25391391 Gomes TAT, Toledo MRF, Trabulsi LR, Wood PK, Morris JG. DNA probes for identification of enteroinvasive Escherichia coli. J Clin Microbiol. 1987;25:2025-2027.; however, these are sporadic cases. Recently, the serotype of E. coli O96:H19 was described as enteroinvasive E. coli in two large outbreaks occurring in Italy and United Kingdom.392392 Michelacci V, Prosseda G, Maugliani A, et al. Characterization of an emergent clone of enteroinvasive Escherichia coli circulating in Europe. Clin Microbiol Infect. 2016;22(3), 287.e11-9.,393393 Newitt S, MacGregor V, Robbins V, et al. Two linked enteroinvasive Escherichia coli outbreaks, Nottingham, UK, June 2014. Emerg Infect Dis. 2016;22(7):1178-1184. It is worth mentioning that EIEC serotypes considered to be nonmotile produce an unusually large (77 kDa) flagellin that is assembled into functional flagellum filaments that allow the bacteria to swim in modified motility agar (0.2%).394394 Andrade A, Girón JA, Amhaz JMK, Trabulsi LR, Martinez MB. Expression and characterization of flagella in nonmotile enteroinvasive Escherichia coli isolated from diarrhea cases. Infect Immun. 2002;70:5882-5886. Analysis of the fliC gene showed that 11 different EIEC serotypes have six molecular profiles of fliC. The major EIEC serotypes showed low fliC diversity. The dendrogram showed two major clusters, suggesting two different origins for the flagellin gene among these strains. In addition, the presence of the same pattern among strains of the same serotype suggests the existence of a common clone.395395 Amhaz JMK, Andrade A, Bando SY, Tanaka TL, Moreira-Filho CA, Martinez MB. Molecular typing and phylogenetic analysis of enteroinvasive Escherichia coli using the fliC gene sequence. FEMS Microbiol Lett. 2004;235:259-264.

Virulence factors, mechanisms and pathogenesis

Diarrhea due to EIEC and Shigella is caused by the invasion and penetration of bacteria in the enterocytes, leading to their destruction. These bacteria bind specifically to the mucosa of the large intestine and invade cells by endocytosis.396396 Levine MM. E. coli that cause diarrhea enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorragic and enteroaderent. J Infect Dis. 1987;155:377-389.,397397 Sansonetti PJ, Kopecko DJ, Formal SB. Involvement of a palsmid in the invasive ability of Shigella flexneri. Infect Immun. 1982;35:852-860.Shigella flexneri strains are used as template for most studies of invasion.

The complex process in colonization and EIEC survival in the gastrointestinal barrier depends on the presence of a large plasmid of about 220 kb (pInv), very similar to that found in Shigella.397397 Sansonetti PJ, Kopecko DJ, Formal SB. Involvement of a palsmid in the invasive ability of Shigella flexneri. Infect Immun. 1982;35:852-860.

398 Formal SB, Hale TL, Sansonetti PJ. Invasive enteric pathogens. Rev Infect Dis. 1983;5:702-707.

399 Harris JR, Wachsmuth IK, Davis BR, Cohen ML. High-molecular-weight plasmid correlates with Escherichia coli enteroinvasiveness. Infect Immun. 1982;37:1295-1298.
-400400 Gibotti A, Tanaka TL, Oliveira VR, Taddei CR, Martinez MB. Molecular characterization of enteroinvasive Escherichia coli ipa genes by PCR-RFLP analysis. Braz J Microbiol. 2004;35:74-80. In this process, multiple bacterial genes are involved, both chromosomal and plasmidial. Bacteria without the virulence plasmid do not cause keratoconjunctivitis in guinea pigs, being considered non-virulent.397397 Sansonetti PJ, Kopecko DJ, Formal SB. Involvement of a palsmid in the invasive ability of Shigella flexneri. Infect Immun. 1982;35:852-860.,401401 Small PLC, Isberg RR, Falkow S. Comparation of the ability of enteroinvasive E.coli, Salmonella typhimuriun, Y. pseudotuberculosis, and Y. enterocolitica to enter and replicate within Hep-2 cells. Infect Immun. 1987;55:1674-1679.

Most of these functions are related to proteins encoded by a 31-kb fragment from pInv, containing 38 genes. In this fragment are genes responsible for bacterial invasion and escape, by cell spreading, inhibition of autophagy, regulation of immune response of the host apparatus and type III secretion system (TTSS). Once injected into the host cell, the virulence or effector factors induce or inhibit cell signaling pathways. The changes in host cells induced by bacteria allow intracellular survival of these microorganisms.402402 Cossart P, Sansonetti PJ. Bacterial invasion: the paradigm of enteroinvasive pathogens. Science. 2004;304:242-248.

403 Parsot C. Shigella spp. and enteroinvasive Escherichia coli pathogenicity factors. FEMS Microbiol Lett. 2005;252:11-18.
-404404 Ogawa M, Handa Y, Ashida H, Suzuki M, Sasakawa C. The versatility of Shigella effectors. Nat Rev Microbiol. 2008;6:11-16.

Due to the great similarity between Shigella and EIEC, it can be assumed that the two would share the same ancestor and that at a given moment in evolution there was a division. Why has EIEC retained some E. coli properties that have been lost in multiple lineages of Shigella? Data obtained by different groups lead to the speculation that EIEC strains are in an intermediate stage and are a potential pre-cursor of “full-blown” Shigella strains.405405 Bando SY, Valle GRF, Martinez MB, Trabulsi LR, Moureira-Filho CA. Characterization of enteroinvasive Escherichia coli and Shigella strains by RAPD analysis. FEMS Microbiol Lett. 1998;165:159-165.

406 Lan R, Alles MC, Donohoe K, Martinez MB, Reeves PR. Molecular evolutionary relationships of enteroinvasive Escherichia coli and Shigella spp. Infect Immun. 2004;72(9):5080-5088.

407 Martinez MB, Whittan TS, McGraw EA, Rodrigues J, Trabulsi LR. Clonal relationship among invasive and non-invasive strains of enteroinvasive Escherichia coli serogroups. FEMS Microbiol Lett. 1999;172(2):145-151.

408 Pupo GM, Karaolis DKR, Lan R, Reeves PR. Evolutionary relationships among pathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies. Infect Immun. 1997;65:2685-2692.
-409409 Rolland K, Lambert-Zechovsky N, Picard B, Denamur E. Shigella and enteroinvasive Escherichia coli strains are derived from distinct ancestral strains of E. coli. Microbiology. 1998;144(9):2667-2672.

Despite the similarities invasion mechanism and symptoms of the disease (dysentery), the infectious dose of EIEC is much higher than that of Shigella.410410 Dupont HL, Formal SB, Hornick RB, et al. Phatogenesis of Escherichia coli diarrhea. N Engl J Med. 1971;285:1-9. Furthermore, the disease caused by EIEC appears to be a milder and self-limiting form.

In the Serény test, it was observed that EIEC induces a milder form of the disease (mild/moderate inflammation), while Shigella leads to an exacerbation of proinflammatory response (severe inflammation). Furthermore, keratoconjunctivitis develops more rapidly in guinea pigs inoculated with Shigella (two days) than in guinea pigs inoculated with EIEC (4-5 days).411411 Moreno AC, Ferreira LG, Martinez MB. Enteroinvasive Escherichia coli vs. Shigella flexneri: how different patterns of gene expression affect virulence. FEMS Microbiol Lett. 2009;301:156-163.

Samples from different serotypes of EIEC have shown polymorphism in some regions of genes involved in invasion. However, the data reveal that there are no changes in genes of the invasion plasmid antigens that could explain the differences in pathogenicity between Shigella and EIEC.400400 Gibotti A, Tanaka TL, Oliveira VR, Taddei CR, Martinez MB. Molecular characterization of enteroinvasive Escherichia coli ipa genes by PCR-RFLP analysis. Braz J Microbiol. 2004;35:74-80. Moreover, recent studies from our group showed that the genes responsible for cell spreading (icsA and icsB) and regulation of the immune response of the host (osp) did not indicate changes that could explain the difference in pathogenicity between Shigella and EIEC (data not shown).

Another important aspect of bacterial colonization is the uptake of iron (Fe) under conditions limited in the host. Iron is an essential element for all living organisms, it is estimated that the microorganisms require iron at concentrations from 10 to 10−6 M to meet their metabolic needs. It was shown that EIEC has a high adaptability, using, if necessary, the iron capture system that consumes less energy. The ability to capture Fe from different sources can facilitate the development of infectious processes by this bacterium.412412 Andrade A, Dall’Agnol M, Newton S, Martinez MB. The iron uptake mechanisms of enteroinvasive Escherichia coli. Braz J Microbiol. 2000;31:200-205.,413413 Dall’Agnol M, Martinez MB. Iron uptake from host compounds by enteroinvasive Escherichia coli. Rev Microbiol. 1999;30:149-152.

EIEC, like other enteric pathogens, target M cells (microfold cells) present in the intestinal mucosa as a route of entry to deeper tissues of the host.403403 Parsot C. Shigella spp. and enteroinvasive Escherichia coli pathogenicity factors. FEMS Microbiol Lett. 2005;252:11-18.,414414 Sansonetti PJ, Phalipon AM. Cells as ports of entry for enteroinvasive pathogens: mechanisms of interaction, consequences for the disease process. Semin Immunol. 1999;11:193-203. Reaching the lamina through the M cells, the bacterial cells are phagocytized by macrophages and dendritic cells. These cells are the first step in the production of the inflammatory response against bacterial invasion. After escape from macrophages and dendritic cells, EIEC are able to invade enterocyte cells from the basolateral side, escaping from the phagosome and replicating in the cytoplasm.403403 Parsot C. Shigella spp. and enteroinvasive Escherichia coli pathogenicity factors. FEMS Microbiol Lett. 2005;252:11-18.,414414 Sansonetti PJ, Phalipon AM. Cells as ports of entry for enteroinvasive pathogens: mechanisms of interaction, consequences for the disease process. Semin Immunol. 1999;11:193-203.

Our group first described the phenotypic and genotypic characteristics explaining the lower capacity of EIEC to cause disease when compared with the species of Shigella. To this end, use has been made of experimental models that mimick the intestinal microenvironment of the host, such as cultures of intestinal epithelial cells, macrophages and dendritic cells.411411 Moreno AC, Ferreira LG, Martinez MB. Enteroinvasive Escherichia coli vs. Shigella flexneri: how different patterns of gene expression affect virulence. FEMS Microbiol Lett. 2009;301:156-163.,415415 Bando SY, Moreno ACR, Albuquerque JAT, Amhaz JMK, Moureira-Filho CA, Martinez MB. Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study. Mem Inst Oswaldo Cruz. 2010;105(6):1-6.,416416 Moreno AC, Ferreira KS, Ferreira LG, Almeida SR, Martinez MB. Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states. Mem Inst Oswaldo Cruz. 2012;107(1):138-141. Our results showed that the initial ability to invade the intestinal cell is similar between EIEC and Shigella, but that the expression of virulence genes (ipaABCD, icsA, icsB, virF, virB), capacity to escape from the phagosome, intracellular proliferation and dissemination of EIEC, as well as the ability to cause cell damage during the infection, are much lower than with S. flexneri.411411 Moreno AC, Ferreira LG, Martinez MB. Enteroinvasive Escherichia coli vs. Shigella flexneri: how different patterns of gene expression affect virulence. FEMS Microbiol Lett. 2009;301:156-163. A significantly greater number of EIEC are seen inside macrophages compared to Shigella after phagocytosis. Furthermore, Shigella shows greater capacity to escape from macrophages as compared to EIEC. The expression of virulence genes, production of proinflammatory cytokines and cell death was found to be less in macrophages infected by EIEC when compared to Shigella. It should be noted that the production of antiinflammatory cytokine IL-10 by macrophages is greater in infection by EIEC than Shigella.415415 Bando SY, Moreno ACR, Albuquerque JAT, Amhaz JMK, Moureira-Filho CA, Martinez MB. Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study. Mem Inst Oswaldo Cruz. 2010;105(6):1-6.

EIEC interaction with dendritic cells has been evaluated. The data suggest that EIEC induces the production of IL-10, IL-12 and TNF-α by infected dendritic cells, while S. flexneri induce TNF-α production. Unlike Shigella, infection with EIEC increases the expression of TLR-4 and TLR-5 receptors on dendritic cells and decreases the expression of costimulatory molecules that may cooperate to induce the proliferation of T-lymphocytes, and in addition, there is a greater proliferation of lymphocytes challenged with S. flexneri than with EIEC.416416 Moreno AC, Ferreira KS, Ferreira LG, Almeida SR, Martinez MB. Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states. Mem Inst Oswaldo Cruz. 2012;107(1):138-141.

Epidemiology

The EIEC strains have similar biochemical, genetic and pathogenic characteristics as Shigella species, which can often make the correct identification of this pathotype difficult.381381 Silva RM, Toledo MRF, Trabulsi LR. Biochemical and cultural characteristics of invasive Escherichia coli. J Clin Microbiol. 1980;11:441-444.,385385 Trabulsi LR, Fernandes MRF. Antigenic identity of culture 193T-64 and E.coli O136:K78 (B22). Rev Inst Med Trop. 1969;11:101-103.,386386 Sakazaki R, Tamura K, Saito M. Enteropathogenic Escherichia coli associated with diarrhea in children and adults. Jpn J Med Sci Biol. 1967;20:387-399.,417417 Trabulsi LR, Fernandes MRF, Zuliani ME. Novas bactérias patogênicas para o intestino do homem. Rev Inst Med Trop. 1967;9:31-39. Epidemiological data may be underestimated due to the difficulty in differentiating between Shigella and EIEC.

EIEC was responsible for several outbreaks, but there are few reports on routes of transmission and distribution of this bacterium in nature. Water and cheese were described as potential sources,418418 Borian A, Csizmazia F, Karvaly E, Mihalffy F, Redey B. Enterocolitis epidemic caused by water contamined with E. coli O124 in Veszpren. Orv Hetil. 1959;100:1072-1074.

419 Marier R, Wells JC, Swanson RC, Callahan W, Mehlman IJ. An outbreak of enteropathogenic E. coli foodborne disease traced to imported cheese. Lancet. 1973;302:1376-1378.

420 Tulloch JEF, Ryan KJ, Formal SB, Franklin FA. Invasive enterophatic Escherichia coli dysentery. Ann Intern Med. 1973;79:13-17.
-421421 Valentini SR, Gomes TAT, Falcão DP. Lack of virulence factors in Escherichia coli strains of enteropathogenic serougroups isolated from water. Appl Environ Microbiol. 1992;58:412-414. as well as the direct transmission through person-to-person contact.422422 Harris JR, Mariano J, Wells JG, Payne BJ, Donnell HD, Cohen ML. Person-to-person transmission in an outbreak of enteroinvasive Escherichia coli. Am J Epidemiol. 1985;122:245-252. In the 1970s, a major outbreak of diarrhea was reported in the United States, which affected 387 patients. The transmission vehicle was an imported cheese, contaminated by O124 serogroup.419419 Marier R, Wells JC, Swanson RC, Callahan W, Mehlman IJ. An outbreak of enteropathogenic E. coli foodborne disease traced to imported cheese. Lancet. 1973;302:1376-1378. According to the food and drug control agency of the United States (Food and Drug Administration-FDA), outbreaks caused by EIEC have been associated with milk and milk products and beef; however, any food or water contaminated with human feces of an individual patient can cause disease in other individuals.423423 Food and Drug Administration. Enteroinvasive Escherichia coli (EIEC); 2009. http://www.fda.gov/Food/FoodSafety/FoodborneIllness/FoodborneIllnessFoodbornePathogensNaturalToxins/BadBugBook/ucm071298.htm.
http://www.fda.gov/Food/FoodSafety/Foodb...
In Brazil, there is a report of three samples isolated from water.421421 Valentini SR, Gomes TAT, Falcão DP. Lack of virulence factors in Escherichia coli strains of enteropathogenic serougroups isolated from water. Appl Environ Microbiol. 1992;58:412-414. Outbreaks involving two EIEC were recently reported in Europe, one in Italy in 2012 involving 109 cases and another in the United Kingdom in 2014 involving 50 cases.392392 Michelacci V, Prosseda G, Maugliani A, et al. Characterization of an emergent clone of enteroinvasive Escherichia coli circulating in Europe. Clin Microbiol Infect. 2016;22(3), 287.e11-9.,393393 Newitt S, MacGregor V, Robbins V, et al. Two linked enteroinvasive Escherichia coli outbreaks, Nottingham, UK, June 2014. Emerg Infect Dis. 2016;22(7):1178-1184. In both, vegetables were to blame.

In Calcutta, the prevalence of EIEC in a group of 263 patients hospitalized with diarrhea was high, 16.3% of cases.424424 Chaterjee BD, Sanyal SN. Is it all shigellosis? Lancet. 1984;2:574. However, there are reports of a prevalence of 2%.425425 Ram S, Khurana S, Khurana SB, Sharma S, Vadehra DV. Seasonal fluctuations in the occurrence of enteroinvasive Escherichia coli diarrhea. Indian J Med Res Sec A. 1990;91:258-262. In Thailand, China and other Asian countries, a prevalence of 4 to 7% has been seen.426426 Echeverria P, Sethabutr O, Serichantalergs O, Lexomboon U, Tamura K. Shigella and enteroinvasive Escherichia coli infections in households of children with dysentery in Bangkok. J Infect Dis. 1992;165:144-147.

427 Kain KC, Barteluk RL, Kelly MT, et al. Etiology of childhood diarrhea in Beijing, China. J Clin Microbiol. 1991;29:90-95.

428 Tamura K, Sakazaki R, Murase M, Kosako Y. Serotyping and categorisation of Escherichia coli strains isolated between 1958 and 1992 from diarrhoeal diseses in Asia. J Med Microbiol. 1996;45:353-358.
-429429 Taylor DN, Escheverria P, Pál T, et al. The role of Shigella sp, enteroinvasive Escherichia coli, and other enteropathogens as causes of childhood dysentery in Thailand. J Infect Dis. 1986;153:1132-1138. In Bolivia, the reports showed a 2% prevalence.430430 Utsunomiya A, Elio-Calvo D, Reyes AA, et al. Major enteropathogenic bacteria isolated from diarrheal patients in Bolivia: a hospital-based study. Microbiol Immunol. 1995;39:845-851. Some studies have shown that in Nigeria, Iran and Thailand, the distribution of EIEC is below (less than 0.1%) the rates found in developed countries; in Spain, for example, a prevalence of 0.2% was found.431431 Katouli M, Jaari A, Moghaddam AA, Ketabi GR. Etiological studies of diarrheal diseases in infants and young children in Iran. J Trop Med Hyg. 1990;93:22-27.

432 Ogunsanya TI, Rotimi VO, Adenuga A. A study of aetiological agents of childhood diarrhoea in Lagos, Nigeria. J Med Microbiol. 1994;40:10-14.

433 Prats G, Llovet T. Enteroinvasive Escherichia coli: pathogenic mechanisms and epidemiology. Microbiologia. 1995;11:91-96.
-434434 Sunthadvanich R, Chiewsilp D, Seriwatana J, Sakazaki R, Echeverria P. Nation wide surveillance program to identify diarrhea-causing Escherichia coli in children in Thailand. J Clin Microbiol. 1990;28:469-472. The low incidence can be due to difficulties in differentiating EIEC from Shigella.

The isolation of EIEC in Brazil has ranged from 0.5 to 15%, depending on the population investigated.435435 Almeida MTG, Silva RM, Donaire LM, Moreira LE, Martinez MB. Enteropathogens associated with acute diarreal disease in children. J Pediatr. 1998;74:291-298.

436 Moreno AC, Fernandes-Filho A, Gomes TAT, et al. Etiology of childhood diarrhea in the northeast of Brazil: significant emergent diarrheal pathogens. Diagn Microbiol Infect Dis. 2010;66:50-57.

437 Lozer DM, Souza TB, Monfardini MV, et al. Genotypic and phenotypic analysis of diarrheagenic Escherichia coli strains isolated from Brazilian children living in low socioeconomic level communities. BMC Infect Dis. 2013;13:418.

438 Souza EC, Martinez MB, Taddei CR, et al. Etiology profile of acute diarrhea in children in São Paulo. J Pediatr. 2002;78(1):31-38.

439 Toledo MRF, Trabulsi LR. Frequency of enteroinvasive Escherichia coli in children with diarrhea and healthy controls, in São Paulo, SP, Brazil. Rev Microbiol. 1990;21:1-4.
-440440 Oliveira MG, Pessoa GVA, Nakahara LK. Enteropathogenic bacteria occurrence in diarrheic children living in Juiz de Fora municipality Minas Gerais Brazil. Rev Inst Adolfo Lutz. 1989;49:161-168. The data suggest that the presence of EIEC is related to socioeconomic conditions. Toledo and Trabulsi439439 Toledo MRF, Trabulsi LR. Frequency of enteroinvasive Escherichia coli in children with diarrhea and healthy controls, in São Paulo, SP, Brazil. Rev Microbiol. 1990;21:1-4. investigated the presence of this microorganism from children under five years of age and non-slum-dwelling children from different areas of the city of São Paulo. This bacterium has been found in 17 of 107 slum-dwelling children with diarrhea (15.9%) and in 16 of 701 non-slum-dwelling children with diarrhea (2.3%). In the first group, EIEC was the enteropathogen most frequently isolated from children over 2 years of age. In non-slum-dwelling children of the same age, it was the fourth most common agent, being more frequent than EPEC, Salmonella, Rotavirus, and Yersinia enterocolitica. Studies performed outside the city of São Paulo showed a low prevalence of these bacteria, 0.5-2.5%.435435 Almeida MTG, Silva RM, Donaire LM, Moreira LE, Martinez MB. Enteropathogens associated with acute diarreal disease in children. J Pediatr. 1998;74:291-298.,440440 Oliveira MG, Pessoa GVA, Nakahara LK. Enteropathogenic bacteria occurrence in diarrheic children living in Juiz de Fora municipality Minas Gerais Brazil. Rev Inst Adolfo Lutz. 1989;49:161-168.

Detection and diagnosis

Samples of EIEC grow well in culture medium routinely used for isolation of Enterobacteriaceae, such as MacConkey agar, xylose-lysine-deoxycholate (XLD) agar and Hektoen enteric (HE). Highly selective media such as Salmonella Shigella agar (SS) or bismuth sulfite agar may not be as effective for some serotypes.381381 Silva RM, Toledo MRF, Trabulsi LR. Biochemical and cultural characteristics of invasive Escherichia coli. J Clin Microbiol. 1980;11:441-444.

The identification of E. coli species may be carried out using conventional biochemical tests, such as production of indole, fermentation of glucose, sucrose and lactose, gas production from glucose fermentation, pathway glucose fermentation, using citrate as sole carbon source, motility, lysine, arginine and ornithine decarboxylation.11 Ewing WH. Edwards and Ewing's Identification of Enterobacteriaceae. 4th ed. New York: Elsevier; 1986.,441441 Toledo MRF, Trabulsi LR. Correlation between biochemical and serological characteristics of Escherichia coli and results of the Sereny Test. J Clin Microbiol. 1983;17(3):419–421. Fermentation of lactose varies according to the strain; EIEC samples can ferment lactose slowly (72 h), making it difficult to differentiate from Shigella.381381 Silva RM, Toledo MRF, Trabulsi LR. Biochemical and cultural characteristics of invasive Escherichia coli. J Clin Microbiol. 1980;11:441-444. Complementary to the physiological and biochemical characteristics, serotyping may be required for the differentiation, since some serotypes of S. flexneri produce indole. In such cases, O antisera of EIEC and Shigella should be used.11 Ewing WH. Edwards and Ewing's Identification of Enterobacteriaceae. 4th ed. New York: Elsevier; 1986.,441441 Toledo MRF, Trabulsi LR. Correlation between biochemical and serological characteristics of Escherichia coli and results of the Sereny Test. J Clin Microbiol. 1983;17(3):419–421. Bacterial colonies with this characteristic can be screened for the classical EIEC serogroups O28ac, O29, O112, O124, O136, O143, O144, O152, O159, O164, O169, and O173.11 Ewing WH. Edwards and Ewing's Identification of Enterobacteriaceae. 4th ed. New York: Elsevier; 1986.,441441 Toledo MRF, Trabulsi LR. Correlation between biochemical and serological characteristics of Escherichia coli and results of the Sereny Test. J Clin Microbiol. 1983;17(3):419–421.,443443 Ørskov F, Ørskov I. Escherichia coli serotyping and disease in man and animals. Can J Microbiol. 1992;38:699-704. EIEC invasive capacity can be evaluated using the Sereny guinea pig eye test444444 Sereny B. Experimental Shigella keratoconjunctivitis. A preliminary report. Acta Microbiol Acad Sci Hung. 1955;2:293-296. and tissue culture assays,445445 Oaks EV, Hale TL, Formal SB. Serum immune response to Shigella proteins antigens in rhesus monkeys and human infected with Shigella spp. Infect Immun. 1986;53:57–63. which are more markedly limited to reference laboratories.

To characterize the EIEC pathotype, it is necessary to search for plasmid virulence genes. Currently, the investigation of the ipaH gene, a multi-copy gene (4-10) present in EIEC and Shigella, by PCR is recommended,442442 Ud-Din A, Wahid S. Relationship among Shigella spp. and enteroinvasive Escherichia coli (EIEC) and their differentiation. Braz J Microbiol. 2014;45(4):1131-1138.,446446 Pavlovic M, Luze A, Konrad R, et al. Development of a duplex real-time PCR for differentiation between E. coli and Shigella spp. J Appl Microbiol. 2011;110:1245-1251. or studies of other DNA sequences are needed, such as the invasion-associated locus gene (ial).447447 Escher M, Scavia G, Morabito S, et al. A severe foodborne outbreak of diarrhoea linked to a canteen in Italy caused by enteroinvasive Escherichia coli, an uncommon agent. Epidemiol Infect. 2014;142:2559-2566. The presence of the iudA and lacY genes can differentiate EIEC from S. flexneri.446446 Pavlovic M, Luze A, Konrad R, et al. Development of a duplex real-time PCR for differentiation between E. coli and Shigella spp. J Appl Microbiol. 2011;110:1245-1251. A simple and rapid stool test based on apyrase (ATP-diphosphohydrolase) activity was described for EIEC detection.448448 Sankaran K, Banerjee S, Pavankumar AR, Jesudason M, Reissbrodt R, Williams PH. Apyrase-based colorimetric test for detection of Shigella and enteroinvasive Escherichia coli in stool. Diagn Microbiol Infect Dis. 2009;63:243-250. This is an essential periplasmic enzyme required for unipolar localization of IcsA, which is involved in the pathogen's intracellular and intercellular spread, and is only expressed by EIEC and Shigella.449449 Bhargava T, Datta S, Ramachandran R, Roy RK, Sankaran K, Subrahmanyam YVBK. Virulent Shigella codes for a soluble apyrase: identification, characterization and cloning of the gene. Curr Sci. 1995;68:293-300. The enzyme activity is measured by a colorimetric reaction. The method is robust, requires widely available equipment and affordable reagents, and can be applied for routine use in laboratories with limited resources.448448 Sankaran K, Banerjee S, Pavankumar AR, Jesudason M, Reissbrodt R, Williams PH. Apyrase-based colorimetric test for detection of Shigella and enteroinvasive Escherichia coli in stool. Diagn Microbiol Infect Dis. 2009;63:243-250.

Conclusions

The genomic plasticity of E. coli strains is noteworthy, as can be seen by the variety of strains ranging from commensal residents of the gastrointestinal tract to assorted pathogens that are able to promote intestinal or extraintestinal illnesses with different clinical consequences. It is thus important to note that the continuous evolution of the E. coli genome has hindered the classification of certain E. coli isolates into a pathotype, because some isolates combine the main virulence characteristics of different pathotypes and are thus considered hybrid pathotypes (reviewed in 55 Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26(4):822-880.) with the potential of allowing the rise of new and more virulent pathogenic E. coli hybrids.

Whole-genome sequencing has provided a great amount of useful information on the genome of pathogenic E. coli, which will help improve diagnosis, typing, disease management, epidemiology and outbreak investigations as well as helping to monitor the spread of pathogens.55 Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26(4):822-880. Despite the recent advances in our knowledge of the genetic background and pathogenicity of strains of different DEC pathotypes, various novel genes encoding unknown functions are yet to be characterized to further our understanding of the interactions of these pathogens with their hosts.

Acknowledgement

The authors dedicate the present article to Prof. Luiz R. Trabulsi who, during his life, inspired us and several others in the study of microbiology, particularly in E. coli pathogenesis. He also left to all who met him in life a wonderful and unforgettable example of professional dedication and ethical commitment in science and education.

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Publication Dates

  • Publication in this collection
    Dec 2016

History

  • Received
    08 Oct 2016
  • Accepted
    27 Oct 2016
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br