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Epidemiology and Clinical Manifestations of EnteroaggregativeEscherichia coli
Betina Hebbelstrup Jensen,a Katharina E. P. Olsen,a Carsten Struve,a Karen Angeliki Krogfelt,a Andreas Munk Petersena,b,c
Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmarka; Department of Gastroenterology, Hvidovre Hospital, Copenhagen,Denmarkb; Department of Clinical Microbiology, Hvidovre Hospital, Copenhagen, Denmarkc
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .614INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .614PATHOGENICITY OF EAEC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .615
Adherence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .615Biofilm Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .615Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .615Pathogenicity Islands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .616Phylogeny of EAEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .616Volunteer Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .616
DIAGNOSIS OF EAEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .617CLINICAL MANIFESTATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .617
Inflammatory Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .617Host Factors Are Determinants of EAEC Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .620Immunological Response to EAEC Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .620
MULTIDRUG RESISTANCE IN EAEC STRAINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .620RESERVOIR AND TRANSMISSION OF EAEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .620EAEC AS A CAUSE OF CHILDHOOD DIARRHEA IN DEVELOPING COUNTRIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .621ASSOCIATION BETWEEN EAEC AND HIV-RELATED DIARRHEA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .621TRAVELER’S DIARRHEA AND EAEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .622ASSOCIATION BETWEEN EAEC AND DIARRHEA IN INDUSTRIALIZED COUNTRIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .622URINARY TRACT INFECTIONS AND EAEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .623OUTBREAKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .623
The German O104:H4 EAEC Outbreak in 2011. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .624DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .624CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .625ACKNOWLEDGMENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .625REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .625AUTHOR BIOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .630
SUMMARY
Enteroaggregative Escherichia coli (EAEC) represents a heteroge-neous group of E. coli strains. The pathogenicity and clinical rele-vance of these bacteria are still controversial. In this review, wedescribe the clinical significance of EAEC regarding patterns ofinfection in humans, transmission, reservoirs, and symptoms.Manifestations associated with EAEC infection include watery di-arrhea, mucoid diarrhea, low-grade fever, nausea, tenesmus, andborborygmi. In early studies, EAEC was considered to be an op-portunistic pathogen associated with diarrhea in HIV patients andin malnourished children in developing countries. In recent stud-ies, associations with traveler’s diarrhea, the occurrence of diar-rhea cases in industrialized countries, and outbreaks of diarrhea inEurope and Asia have been reported. In the spring of 2011, a largeoutbreak of hemolytic-uremic syndrome (HUS) and hemorrhagiccolitis occurred in Germany due to an EAEC O104:H4 strain,causing 54 deaths and 855 cases of HUS. This strain producesthe potent Shiga toxin along with the aggregative fimbriae. Anoutbreak of urinary tract infection associated with EAEC in Co-penhagen, Denmark, occurred in 1991; this involved extensiveproduction of biofilm, an important characteristic of the patho-genicity of EAEC. However, the heterogeneity of EAEC continues
to complicate diagnostics and also our understanding of pathoge-nicity.
INTRODUCTION
Diarrhea is still an important disease burden worldwide. Itcauses considerable childhood mortality in the developing
world and is associated with morbidity and substantial health carecosts in industrialized countries (1). One important cause of in-fectious diarrhea is the so-called diarrheagenic Escherichia coli(DEC) group (1, 2). The following subgroups of DEC have beendefined: enterotoxigenic E. coli (ETEC), Shiga toxin-producing E.coli (STEC), enteroinvasive E. coli (EIEC), enteropathogenic E.coli (EPEC), and enteroaggregative E. coli (EAEC) (3–5). Recently,the pathogenicity of these pathotypes, including EAEC, was re-viewed (5); in this review, we focus on the epidemiology and dis-ease manifestations of EAEC specifically. EAEC was first describedin 1987 by Nataro et al. in a study examining different patterns of
Address correspondence to Karen Angeliki Krogfelt, [email protected].
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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adherence of E. coli strains to HEp-2 cells in culture. The strainswere collected from Chilean children with diarrhea, and the typi-cal “stacked-brick” pattern was observed (6), which continues todefine EAEC (Fig. 1). The aggregative adherence (AA) pattern isdefined as the binding of bacteria to epithelial cells in a stacked-brick manner. Although EAEC has been associated with diarrheain studies conducted in both developing and industrialized coun-tries, it has been difficult to determine the specific mechanisms ofEAEC pathogenicity, which has made assessments of the clinicalrelevance of this microorganism difficult. Here, we present theliterature on this subject, concentrating on the pathophysiology ofEAEC.
PATHOGENICITY OF EAEC
Regarding infection with any of the subtypes of EAEC strains, thefollowing stages of pathogenesis have been described: (i) initialadherence to the mucosal surface, (ii) biofilm formation, and (iii)induction of an inflammatory response and release of toxins (7–12) (Fig. 2). Knowledge of the stages of EAEC pathogenicity hasbeen obtained from studies of in vitro cell cultures, animal models,and patients infected with EAEC (10, 13–17).
Adherence
Adhesion to the intestinal epithelium is facilitated by fimbriae andis the first step in the bacterial colonization of the gut (10, 18, 19)(Table 1). The aggregative adherence fimbriae (AAF) have beenfound to include 4 major variants, with distinct structures of thepilin subunits (AAF/I to AAF/IV) (19–22). The fimbriae can splayout from the bacteria due to the surface protein dispersin (9, 23),which is encoded by the aap gene. Dispersin is believed toinduce changes in the electrostatic surface of the lipopolysac-charide layer of the bacteria. This makes this protein importantfor the adherence properties of EAEC (9). The fimbriae bind tocomponents of the extracellular matrix of intestinal epithelialcells, such as laminin, collagen IV, cytokeratin 8, and fibronec-tin (24, 25). The aggregative pattern of these bacterial strains isthought to emerge from binding to the epithelial cell surfaceand binding to adjacent EAEC bacteria (26). Autoaggregation
contributes to the AA pattern, where adherence to epithelialcells also involves interactions between bacteria (18). The plas-mid-borne aggR gene is another important gene for the patho-genesis and adherence properties of EAEC, where strains pos-sessing the aggR gene are known as “typical EAEC strains” (27).aggR is a transcriptional activator that promotes the expressionof both chromosomal and plasmid-encoded virulence factors,including AAF and dispersin (28).
Biofilm Formation
When the gut has been colonized with EAEC, secretion of exces-sive mucus has been described, which is followed by the formationof biofilm (10, 29, 30). Biofilm formation is an important patho-genicity trait of EAEC and is situated mainly in the colon; how-ever, formation of biofilm in the small intestine has also beenreported (10). Biofilm formation may be an important contribu-tory factor in persistent infection by allowing the bacteria to evadethe local immune system and by preventing the transport of anti-bacterial factors, including antibiotics (31, 32). Assays to quantifybiofilm formation have been suggested as a possible method ofscreening for pathogenic EAEC strains (8). In several studies, ex-pression of AAF has been shown to be essential for biofilm forma-tion by EAEC (20, 24, 33–35). Other factors involved in the for-mation of biofilm include the shf gene, which has proven to beimportant for solid-biofilm production in EAEC reference strain042 (36). The genes yafK and fis are also important for biofilmformation, probably due to their involvement in the regulation ofAAF expression (33). Furthermore, in epidemiological studies,the plasmid-borne aatA gene (37), encoding the dispersin trans-porter; the set1A gene (38); and the aggR gene (39) have beenassociated with biofilm formation.
Toxins
Once the biofilm has been established, further damage to the in-testinal epithelium has been described, which is caused by therelease of bacterial toxins. The secretion of toxins is thought toplay an important role in secretory diarrhea, which is a typicalclinical manifestation of EAEC infection (4, 9, 40). The cytotoxiceffects of EAEC involve the secretion of serine protease autotrans-porters of the Enterobacteriaceae (SPATEs) (41). The SPATEs con-
FIG 2 Stages of pathogenesis of EAEC. Numbers in circles show the pro-gression of EAEC pathogenesis. (1) Agglutination of planktonic EAEC bac-teria. (2) Adherence to the intestinal epithelium and colonization of thegut. (3) Formation of biofilm. (4) Release of bacterial toxins, inducingdamage to the epithelium and increased secretion. (5) Establishment ofadditional biofilm.
FIG 1 Characteristic stacked-brick pattern observed when EAEC is culturedon HEp-2 cells. This pattern is also known as a honeycomb formation, whichemerges due to bacterium-bacterium and bacterium-cell interactions. The re-sult is the aggregation of the bacteria in a stacked-brick manner. (Courtesy ofRie Jønsson, Department of Microbiology and Infection Control, Statens Se-rum Institut, Copenhagen, Denmark; reprinted with permission.)
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stitute a large family of extracellular proteases secreted by Entero-bacteriaceae via the type V secretion system (28). The SPATE genescan be either chromosomal or plasmid borne, and they are orga-nized into 2 phylogenetically different classes: class I SPATEs arecytotoxic to epithelial cells and include proteins encoded by thepet, sigA, and sat genes, whereas class II SPATEs have more diverseeffects and include proteins encoded by the pic and Shigella extra-cellular protease (sepA) genes (42, 43). Pet (plasmid-encodedtoxin) cleaves spectrin in the epithelial cytoskeleton (44), resultingin the deformation and exfoliation of the cell, and is associatedwith mucoid stools (45). Sat (secreted autotransporter toxin) hasbeen shown to cause loosening of cellular tight junctions in kidneycells and vacuolation in both kidney cells and bladder cells (46).Pic (protein involved in intestinal colonization) is a mucinase thatinterferes with the integrity of the mucus membrane and inducesserum resistance and hemagglutination (30, 47). The astA geneencodes EAST1 (EAEC heat-stable enterotoxin), which is not aSPATE and which shares certain functional properties of the en-terotoxin (STa) secreted by ETEC (48). This toxin causes in-creased secretion of chloride and has been associated with secre-tory diarrhea (49, 50). However, EAST1 is not confined to EAECstrains, as it has also been detected in EPEC, ETEC, and EHECstrains (50). Furthermore, the astA gene cannot be detected in allEAEC strains (51–53). The ShET1 enterotoxin encoded by the setgene was first identified in Shigella flexneri and may be associatedwith increased fluid secretion (54). The toxin-induced damageobserved in the intestinal epithelium, associated with EAEC infec-tion, other than that mentioned above includes hemorrhagicnecrosis and shortening of villi, enlarged crypt openings, and for-mation of crypt abscesses (53, 55, 56).
Pathogenicity Islands
Different pathogenicity islands have been identified in EAECstrains. One genomic island is inserted at the tRNA pheU locusand encodes the aaiC-associated type VI secretion system, whichis regulated by the aggR gene (28). The Shigella species she patho-genicity island found in some EAEC strains encodes the SPATEsPic and ShET1 enterotoxin, thereby conferring toxic and mucin-olytic activities (47). Two pathogenicity islands associated withextraintestinal E. coli strains, the Yersinia high-pathogenicity is-land, encoding the yersiniabactin siderophore, and the hly patho-
genicity island, encoding hemolysin and P-fimbriae, have alsobeen found in EAEC isolates (57).
Phylogeny of EAEC
Phylogenetic analysis of E. coli pathotypes segregates the strainsinto 6 major groups, groups A, B1, B2, C, D, and E (58–61).Extraintestinal E. coli strains belong largely to groups B2 and D,whereas commensal E. coli strains frequently belong to group A (5,61). One study investigated the phylogeny of 67 diarrheagenic E.coli strains, including 10 EAEC strains and 31 commensal E. colistrains. EHEC, Shigella species, and ETEC were found to belong togroups A, B1, C, and E, whereas EAEC strains were found to bescattered among all phylogenetic groups investigated (60). An-other study investigating EAEC phylogeny by multilocus enzymeelectrophoresis (MLEE) found 2 clusters of DEC containingEAEC strains. However, other pathotypes were found to fall intothe same clusters on the phylogenetic map (59). One epidemio-logical study investigated the potential clustering of EAEC strainsinto different phylogenetic groups but found strains in phylo-groups A, B1, B2, and D, and the authors of this study concludedthat EAEC originates from multiple lineages (62). The above-de-scribed studies indicate that EAEC strains are phylogeneticallydiverse and do not belong to specific phylogroups.
Volunteer Studies
EAEC as a gastrointestinal pathogen was investigated in a volun-teer study performed by Nataro et al. in 1995 (63). To identifypathogenic EAEC isolates, 4 groups of 5 volunteers were each fed1 of 4 different EAEC strains, 042, JM221, 17-2, or 34b, each at adose of 1010 CFU/ml. The strains were collected from patientssuffering from diarrhea in Peru, Mexico, Chile, and India, respec-tively. EAEC serotype O44:H18 strain 042 expressed AAF/II fim-briae, while strains 17-2, 34b, and JM221 expressed AAF/I fim-briae, which were identified by immunogold electron microscopyand DNA hybridization. EAEC strains 042 and 17-2 were found toexpress the gene encoding the enterotoxin EAST1. Strains JM221and 34b did not express EAST1. EAEC strain 042 caused diarrheain 3 of 5 adults; the 3 other EAEC strains failed to cause diarrhea.An earlier volunteer study (64) found that EAEC serotype O78:H33/35 strain JM221 at a dose of 7 � 108 CFU/ml was associatedwith diarrhea in 2 out of 8 volunteers and at a dose of 1 � 1010
TABLE 1 Key EAEC virulence genesa
Virulence gene(s) Function Location Role in pathogenesis
aggR Transcriptional activator of virulence genes Plasmid AdherenceAAF/I–AAF/IV genes Aggregative adherence fimbriae (4 variants) Plasmid Adherenceaap Antiaggregation protein dispersin Plasmid AdherenceaatA Dispersin transporter protein Plasmid Biofilm productionfis Regulation of AAF expression Chromosome Biofilm productionshf Encoding a Shigella flexneri homolog protein Plasmid Biofilm productionyafK Regulation of AAF expression Chromosome Biofilm productionastA Heat-stable enterotoxin EAST1 Plasmid Toxinspet Plasmid-encoded toxin Plasmid ToxinssepA Shigella extracellular protein Plasmid Toxinssat Secreted autotransporter toxin Chromosome Toxinsset Shigella enterotoxin 1 Chromosome Toxinspic Protein involved in colonization Chromosome Toxinsa The EAEC virulence genes listed are involved in several stages of pathogenicity. The appointed stage of pathogenicity for each virulence gene is given for simplicity.
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CFU/ml was associated with diarrhea in 3 out of 8 volunteers. Theincubation period was shorter with the higher inoculum. The lackof consistency in the pathogenic assessment of the EAEC strains inthe 2 studies may have been be caused by the relatively small studygroups. The numbers of volunteers challenged with strain JM221differed between the studies, with 8 healthy adults being chal-lenged in the study by Mathewson et al. (64) and only 5 volunteersin being challenged in the study performed by Nataro et al. (63).The small numbers of volunteers challenged could have resultedin a misrepresentation of different host factors important forEAEC-induced disease in the two panels of volunteers. As revealedin later studies, host susceptibility is suspected to play a pivotalrole in EAEC pathogenesis, which could partly explain the differ-ent conclusions drawn from the volunteer studies.
DIAGNOSIS OF EAEC
The gold standard for the identification of EAEC remains theHEp-2 cell assay (6, 8, 31, 65). This test is performed only inreference laboratories; it requires cell culture facilities and is time-consuming (66). Molecular techniques have been developed todetect EAEC, of which PCR amplification of specific EAEC viru-lence-associated genes is of great importance. However, the ge-netic heterogeneity of EAEC and the notion that EAEC virulenceprobably results from a combination of multiple factors make it achallenge to differentiate pathogenic and nonpathogenic strainsby the molecular methods used today (31, 51, 67, 68). Variousgene targets have been used to detect EAEC by using PCR (Tables2 and 3). One EAEC-specific gene is the chromosomal aaiC gene,which is a gene in a genomic island encoding a type VI secretionsystem (28). Additional EAEC genes that are frequently used todetect EAEC by PCR include aggR and aatA (11, 65, 69–71).
In the volunteer study by Nataro et al. (63), EAEC strain 042,eliciting the stacked-brick pattern, was shown to cause diarrhea inthe majority of study participants, and since then, it has served asthe main EAEC reference strain. Reference strains representingspecific EAEC genes, for performing PCR and observing the ag-gregative pattern in the HEp-2 cell assay, include 042 (aatA, aggR,aap, and aaiC), JM221 (aggA and sat), 17-2 (aatA and aggR),55989 (agg3A and agg3/4C), and C1010 (agg4A, agg3/4C, sat, andsepA). AAF/I, -II, -III, and -IV are expressed by reference strains17-2, 042, 55989, and C1010, respectively (6, 22, 42, 66, 71, 72)(Table 2).
Serotyping of EAEC strains has proven to be unsuitable due tothe considerable diversity among strains and the large number ofso-called “rough” strains, which are strains that do not express anO antigen (73–76). Autoagglutination caused by the aggregative
phenotype is another obstacle when serotyping EAEC, leading to alarge number of nontypeable strains (23, 26, 42, 77). Serotypingand phylogenetic typing of EAEC have therefore proven to beuseful only in outbreak-related cases (78–82). The typical aggre-gative adherence pattern has been found to be associated with a60-MDa plasmid (pAA) (83). This plasmid carries several viru-lence factors, including the aggregative adherence fimbriae andtoxins (20). A DNA probe named CVD432 (also called the AAprobe) has been constructed from plasmid pAA for the detectionof EAEC by DNA hybridization (83). To identify EAEC, the spec-ificity of the CVD432 probe has been reported to be high but withvarious sensitivities compared to the HEp-2 cell assay (7, 52, 83,84). The lack of sensitivity comes from the genetic heterogeneityof the EAEC strains and the wide geographic dispersal of strainsanalyzed. As suggested in a study by Boisen et al. (20), the combi-nation of virulence genes may depend on the geographic region.International microbiological surveillance of EAEC and an im-proved understanding of EAEC pathogenesis could lead to theachievement of a proper diagnostic algorithm.
CLINICAL MANIFESTATIONS
The symptoms associated with EAEC infections include waterydiarrhea and occasionally very mucoid diarrhea (7, 29, 40), nau-sea, anorexia, low-grade fever, borborygmi, and tenesmus (39, 85,86). Cases of both acute and persistent diarrhea have been de-scribed. Persistent diarrhea is most frequently reported in chil-dren aged �1 year (32, 35, 87, 88). The site of colonization isbelieved to include the colon and the terminal ileum (10, 14, 55,89). The incubation time ranges from 8 h to 52 h (78, 90–92). Astudy by Steiner et al. in 1998 found that children in developingcountries who were diagnosed with EAEC infection suffered fromgrowth retardation regardless of the presence of diarrhea (93). Ina study by Roche et al. (13), growth retardation due to EAECinfection was observed in a mouse model. The growth impairmentwas found to be dependent on the dose of bacteria used for chal-lenge. It was observed that malnourished EAEC-inoculated micehad reduced growth velocity and increased shedding of EAEC instools compared to nourished mice. Bloody diarrhea has beenreported only rarely and involves mostly small children (10, 67,94, 95). However, the German O104:H4 EAEC Shiga toxin-ex-pressing outbreak strain caused hemorrhagic colitis and hemolytic-uremic syndrome (HUS), leading to considerable morbidity and ca-sualties (96–98). The outbreak strain contained the EAEC genes aggR,aggA, set1, pic, and aap and a prophage encoding the stx2 gene (99).Urinary tract infections (UTIs) associated with EAEC (46, 81, 100,101) and one case of urosepsis in an immunosuppressed female (102)have also been described recently. An outbreak of UTIs associatedwith EAEC in 1991 was reported in a Danish study (81), where theUTI outbreak strain contained the following combination of EAECgenes: sat, pic, aatA, aggR, aap, aaiC, and aggA.
Inflammatory Response
The inflammation caused by EAEC has been shown by increasedlevels of fecal interleukin-8 (IL-8), IL-1�, leukocytes, and lacto-ferrin, indicating a substantial gastrointestinal inflammatory re-sponse (11, 87, 103, 104). Occult blood has been detected in stoolsamples from EAEC-positive HIV patients (94). A study bySteiner et al. investigated childhood diarrhea and found increasedlevels of inflammation mediators, even in asymptomatic EAEC-positive carriers, compared to healthy EAEC-negative controls
TABLE 2 EAEC reference strainsa
Referencestrain Gene targets Serotype Reference
042 aatA, aggR, aap, aaiC O44:H18 6JM221 aggA, sat O92:H33 6417-2 aatA, aggR O3:H2 6C-1010 agg4A, agg3/4C, sat, sepA O?:H1 7255989 agg3A, agg3/4C O104:H4 22a Genes, according to the protein encoded, are as follows: aatA, outer membraneprotein; aggR, transcription activator; aap, antiaggregation protein dispersin; aaiC, partof a type VI secretion system; aggA, the major pilin subunit of AAF/I; agg3A, subunit ofAAF/III, sat, secreted autotransporter toxin; agg4A, subunit of AAF/IV; agg3/4C, usher-encoding region from AAF/III and AAF/IV; and sepA, Shigella extracellular protease.
Epidemiology of EAEC
July 2014 Volume 27 Number 3 cmr.asm.org 617
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ownloaded from
TA
BLE
3G
eogr
aph
icdi
stri
buti
onof
EA
EC
case
sa
Ref
eren
ce(s
)Y
rN
o.of
pati
ents
(%E
AE
Cpo
siti
ve)
Cou
ntr
y(ie
s)V
iru
len
cefa
ctor
(s)
and/
ordi
agn
osti
cm
eth
od(s
)St
udy
type
Con
clu
sion
(s)
Val
idit
y
119
1997
798
case
s(2
),58
0co
ntr
ols
(0)
Ger
man
yP
rim
ers
com
plem
enta
ryto
the
CV
D43
2pr
obe
and
HE
p-2
cell
assa
y
Pro
spec
tive
case
-con
trol
EA
EC
was
asso
ciat
edw
ith
wat
ery
diar
rhea
and
chro
nic
diar
rhea
inch
ildre
nSt
ren
gth
sw
ere
larg
est
udy
grou
psan
ddi
arrh
eaco
nfi
rmed
bytr
ain
edpe
rson
nel
;wea
knes
ses
wer
eth
atE
AE
C-
posi
tive
pati
ents
wer
ete
sted
only
for
rota
viru
san
dth
atth
ede
fin
itio
nof
diar
rhea
was
only
2lo
ose
stoo
lsa
day
9319
9818
6(1
4)B
razi
lH
Ep-
2ce
llas
say
and
AA
prob
eC
ohor
tst
udy
EA
EC
-pos
itiv
ech
ildre
nh
adin
crea
sed
leve
lsof
lact
ofer
rin
and
IL-1
�in
stoo
lsam
ples
(P�
0.01
7)an
dsi
gnifi
can
tgr
owth
impa
irm
ent
rega
rdle
ssof
the
pres
ence
ofdi
arrh
ea(P
�0.
05)
Stre
ngt
hs
wer
eth
atit
was
lon
gitu
din
alst
udy
wh
ere
the
nu
trit
ion
alst
atu
san
dgr
owth
rate
sco
uld
beob
serv
edov
erti
me
and
that
stoo
lsam
ples
wer
ete
sted
for
para
site
san
dvi
ruse
s;w
eakn
esse
sw
ere
that
only
1E
.co
liis
olat
ew
asan
alyz
edfu
rth
erpe
rst
ools
ampl
ean
dth
atn
oda
taon
poss
ible
coin
fect
ion
sw
ere
avai
labl
e11
2002
176
case
s(4
9),1
0co
ntr
ols
(0)
Jam
aica
,In
dia,
and
Mex
ico
aggR
,aaf
A,a
ggA
,asp
U,
and
HE
p-2
cell
assa
yD
escr
ipti
ve/c
ase-
con
trol
aggR
-an
daa
fA-p
osit
ive
isol
ates
wer
eas
soci
ated
wit
hin
crea
sed
IL-8
leve
ls(P
�0.
05);
no
asso
ciat
ion
betw
een
AA
F/II
and
diar
rhea
was
obse
rved
Stre
ngt
hs
wer
eth
eu
seof
10h
ealt
hy
con
trol
str
avel
ing
toM
exic
oan
dth
atas
ympt
omat
icE
AE
Cca
rrie
rsw
ere
exam
ined
;wea
knes
ses
wer
eth
atn
oen
teri
cvi
ruse
sw
ere
test
edfo
ran
dth
atth
ere
was
no
men
tion
ofth
epo
ssib
leu
seof
anti
biot
ics
befo
rem
icro
biol
ogic
alte
stin
g69
2005
Neo
nat
es(n
oda
taon
nu
mbe
rs)
Sou
thK
orea
aggA
,aaf
Aag
gR,a
stA
,pic
,pe
t,C
VD
432
prob
e,an
dH
eLa
cell
assa
y
Scre
enin
gSo
me
EA
EC
prob
e-po
siti
veis
olat
esw
ere
not
posi
tive
inth
eH
Ep-
2ce
llas
say;
astA
and
aggR
wer
eth
em
ost
prev
alen
tE
AE
Cvi
rule
nce
gen
es
Stre
ngt
hs
wer
eth
orou
ghm
icro
bial
test
ing
usi
ng
HeL
ace
llas
say,
PFG
E,s
ensi
bilit
yte
stin
g,an
dD
NA
hyb
ridi
zati
on;w
eakn
esse
sw
ere
that
ther
ew
asn
oca
sede
fin
itio
nbe
side
sdi
arrh
eaan
dE
AE
Cst
atu
s,th
ere
was
no
info
rmat
ion
rega
rdin
gth
eu
seof
anti
biot
ics,
stoo
lsa
mpl
esw
ere
not
test
edfo
rga
stro
inte
stin
alvi
ruse
s,th
en
o.of
new
born
ste
sted
was
not
men
tion
ed,a
nd
only
the
no.
ofE
.col
iiso
late
ste
sted
was
men
tion
ed32
2007
122
case
s(5
5),1
27co
ntr
ols
(32)
Bra
zil
aggA
,aaf
Api
c,pe
t,as
tA,
CV
D43
2pr
obe,
and
HeL
ace
llas
say
Cas
e-co
ntr
olT
ypic
alE
AE
Cst
rain
sw
ere
fou
nd
ineq
ual
nu
mbe
rsin
both
grou
ps;t
he
astA
gen
ew
asas
soci
ated
wit
hac
ute
diar
rhea
(P�
0.01
);th
eC
VD
432
prob
ew
asas
soci
ated
wit
hpe
rsis
ten
tdi
arrh
ea(P
�0.
001)
Stre
ngt
hs
wer
eth
eex
clu
sion
ofpa
tien
tsw
ho
had
rece
ived
anti
biot
ics,
thos
ew
ho
had
con
gen
ital
gast
roin
test
inal
mal
form
atio
n,a
nd
thos
ew
ho
had
carb
ohyd
rate
into
lera
nce
and
mat
chin
gby
age
and
soci
oeco
nom
iccl
ass;
wea
knes
ses
wer
eth
atca
ses
wer
epo
orly
defi
ned
as“a
nin
crea
sein
evac
uat
ion
sw
ith
loos
est
ools
”an
dth
atth
ere
was
no
mic
robi
olog
ical
test
ing
for
gast
roin
test
inal
viru
ses
orpa
rasi
tes
inst
ools
ampl
es79
2007
7ca
ses
(71)
Japa
nas
tA,a
ggR
,CV
D43
2pr
obe,
and
HE
p-2
cell
assa
y
Ou
tbre
akA
nE
AE
Cst
rain
ofse
roty
peO
126:
H27
posi
tive
for
the
CV
D43
2pr
obe;
aggR
and
astA
wer
efo
un
din
4pa
tien
tsan
d1
food
han
dler
Stre
ngt
hs
wer
eth
atm
icro
biol
ogic
alte
stin
gin
clu
ded
PC
R,
PFG
E,H
Ep-
2ce
llas
say,
and
sero
typi
ng;
wea
knes
ses
wer
eth
atth
ere
was
no
test
ing
for
gast
roin
test
inal
viru
ses
orpa
rasi
tes
65,7
5,16
020
073,
506
case
s(5
),2,
772
con
trol
s(2
)U
Kaa
tA,a
ap,a
ggR
,agg
C,
agg3
C,a
afC
,shf
,pet
,pi
c,aa
iA,a
stA
,irp
2,ti
a,an
dH
Ep-
2ce
llas
say
Cas
e-co
ntr
olaa
fCw
asth
eon
lyge
ne
asso
ciat
edw
ith
diar
rhea
(P�
0.00
5);a
ggC
was
the
only
gen
eas
soci
ated
wit
hth
eco
ntr
olgr
oup
(P�
0.00
2);E
AE
Cw
asfo
un
dat
ah
igh
freq
uen
cyin
case
san
dco
ntr
ols
Stre
ngt
hs
wer
eth
atth
ere
was
aso
lidca
sede
fin
itio
nan
dre
duce
dbi
asby
excl
udi
ng
pati
ents
wit
hcy
stic
fibr
osis
,C
roh
n’s
dise
ase,
ulc
erat
ive
colit
is,o
rce
liac
dise
ase;
wea
knes
ses
wer
eth
atth
ere
was
no
info
rmat
ion
onpo
ssib
leco
infe
ctio
ns,
no
test
ing
for
gast
roin
test
inal
viru
ses
orpa
rasi
tes,
no
info
rmat
ion
from
stu
dygr
oups
rega
rdin
gtr
avel
,an
dn
oin
form
atio
non
dem
ogra
phic
s15
420
0925
3ca
ses
(7),
751
con
trol
s(3
)U
SAag
gR,a
ggA
,aaf
A,a
ap,
aatA
,ast
A,p
et,s
et1A
,an
dH
Ep-
2ce
llas
say
Cas
e-co
ntr
olst
udy
EA
EC
stra
ins
wit
hag
gR,a
atA
,ast
A,a
ap,a
nd
set1
Aw
ere
asso
ciat
edw
ith
diar
rhea
(P�
0.05
);ag
gR-p
osit
ive
stra
ins
cau
sed
hig
her
leve
lsof
IL-1
ra,I
L-6,
IL-8
,an
dtu
mor
nec
rosi
sfa
ctor
alph
a
Stre
ngt
hs
wer
eth
atth
ere
wer
ela
rge
stu
dygr
oups
,on
lypa
tien
tsw
ith
out
trav
elw
ith
in3
mo
wer
ein
clu
ded,
and
com
mu
nit
y-ac
quir
edE
AE
Cin
fect
ion
sco
uld
bede
tect
ed;w
eakn
esse
sin
clu
ded
ala
ckof
info
rmat
ion
onde
mog
raph
ics
inth
e2
grou
ps,p
osin
ga
poss
ible
sele
ctio
nbi
as78
2008
16ca
ses
(44)
Ital
yaa
p,as
tA,s
et1A
,aat
,agg
R,
and
HE
p-2
cell
assa
yO
utb
reak
An
EA
EC
stra
inw
ith
aggR
,aap
,an
dse
t1A
ofse
roty
peO
92:H
33w
asis
olat
edfr
om6
indi
vidu
als
and
1st
affm
embe
r;su
spec
ted
cau
seof
diar
rhea
was
un
past
euri
zed
chee
se
Stre
ngt
hs
wer
eth
atm
icro
biol
ogic
alte
stin
gof
food
sam
ples
and
stoo
lsam
ples
from
farm
anim
als
was
perf
orm
ed;w
eakn
esse
sw
ere
that
stoo
lsam
ples
wer
ete
sted
for
nor
ovir
us
only
and
not
for
oth
erga
stro
inte
stin
alvi
ruse
s
Hebbelstrup Jensen et al.
618 cmr.asm.org Clinical Microbiology Reviews
on May 20, 2021 by guest
http://cmr.asm
.org/D
ownloaded from
105
2010
170
case
s(8
7),1
04co
ntr
ols
(77)
Sou
ther
nG
han
aaa
tA,a
ap,a
ggR
,an
daa
iCP
rosp
ecti
vecr
oss-
sect
ion
alst
udy
EA
EC
was
asso
ciat
edw
ith
diar
rhea
(P�
0.04
8);E
AE
Cw
asas
soci
ated
wit
hin
crea
sed
feca
llac
tofe
rrin
leve
lsre
gard
less
ofth
epr
esen
ceof
diar
rhea
orn
utr
itio
nal
stat
us
Stre
ngt
hs
wer
eth
eu
seof
aqu
esti
onn
aire
onth
edu
rati
onof
diar
rhea
,dem
ogra
phic
s,liv
ing
con
diti
ons,
brea
stfe
edin
g,an
dco
nsu
mpt
ion
ofm
edic
ine
and
that
anth
ropo
met
ric
mea
sure
men
tsw
ere
incl
ude
din
the
stu
dy;w
eakn
esse
sw
ere
that
the
stoo
lsam
ples
wer
en
otte
sted
for
gast
roin
test
inal
viru
ses
133
2010
394
case
s(1
5),1
98co
ntr
ols
(24)
Sou
thIn
dia
aafI
I,aa
p,as
tA,a
ggR
,AA
prob
eC
ase-
con
trol
EA
EC
was
the
mos
tco
mm
only
isol
ated
DE
Cty
pein
both
grou
ps;t
he
com
bin
atio
nof
the
aap
and
aggR
gen
esan
dth
eA
Apr
obe
was
asso
ciat
edw
ith
diar
rhea
(P�
0.00
1)
Stre
ngt
hs
wer
eth
atch
ildre
nw
ere
test
edfo
rro
tavi
rus
and
Cry
ptos
pori
dium
spec
ies;
wea
knes
ses
wer
eth
atco
infe
ctio
ns
wer
em
enti
oned
,bu
tda
taon
the
dist
ribu
tion
inE
AE
C-p
osit
ive
pati
ents
wer
en
otgi
ven
5220
1014
0(1
1)Ir
anag
gR,a
afA
,agg
Aaa
p,as
tA,
prim
ers
com
plem
enta
ryto
the
CV
D43
2pr
obe,
and
HeL
ace
llas
say
Des
crip
tive
EA
EC
wit
hth
ege
net
icco
mbi
nat
ion
ofag
gR,
aap,
and
astA
was
the
mos
tpr
eval
ent;
only
86.7
%of
PC
R-i
den
tifi
edE
AE
Cba
cter
iaw
ere
posi
tive
inth
eH
eLa
cell
assa
y
Stre
ngt
hs
wer
eth
atin
form
atio
nre
gard
ing
sex,
age,
and
seas
onal
ity
was
avai
labl
e;w
eakn
esse
sw
ere
that
ther
ew
asn
oco
ntr
olgr
oup,
ther
ew
ere
no
data
avai
labl
eon
poss
ible
coin
fect
ion
s,an
dth
ere
was
no
info
rmat
ion
rega
rdin
gre
cen
ttr
avel
1720
1123
ulc
erat
ive
colit
is(6
1),
8C
roh
n’s
dise
ase
(88)
,23
con
trol
s(3
0)
Bra
zil
aggR
,pri
mer
sco
mpl
emen
tary
toth
eC
VD
432
prob
e,an
dH
Ep-
2ce
llas
say
Cas
e-co
ntr
olE
AE
Cw
asfr
equ
entl
yis
olat
edfr
ompa
tien
tssu
ffer
ing
from
Cro
hn
’sdi
seas
ean
du
lcer
ativ
eco
litis
wh
enex
amin
ing
E.c
oli
isol
ates
from
rect
albi
opsy
spec
imen
s
Stre
ngt
hs
wer
eth
ese
lect
ion
ofpa
tien
tsw
ith
wel
l-de
fin
edill
nes
ses
and
info
rmat
ion
onth
eu
seof
anti
biot
ics
and
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Epidemiology of EAEC
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(93). In another study performed in southern Ghana, childrenwho were asymptomatic carriers of EAEC showed increased levelsof fecal lactoferrin (105). These findings suggest that EAEC couldhave pathogenic potential in asymptomatic carriers. The IL-8 in-flammatory response has been shown to be partially caused byflagella (fliC) in a Caco-2 cell assay, as it was found that an aflagel-lar mutant of EAEC did not produce the same inflammatory re-sponse (106).
Host Factors Are Determinants of EAEC Pathogenesis
EAEC has been isolated in high numbers from stool samples ofasymptomatic carriers (35, 107–109), leading to the theory thatthe manifestation of gastrointestinal disease may depend on hostfactors (10, 91, 110). An American study investigated differentsingle nucleotide polymorphisms (SNPs) in the promoter regionof the CD14 gene as predictors of the development of traveler’sdiarrhea due to EAEC (111). The CD14 gene encodes a crucial stepin the inflammatory response to bacterial lipopolysaccharidestimulation by the innate immune system. This study found thatone SNP in the promoter region of the CD14 gene was associatedwith an increased risk of EAEC-induced diarrhea. Patients withthe CD14 �159 TT genotype were significantly associated withEAEC-induced diarrhea (P � 0.008) compared with healthy con-trols. The diverse clinical presentations reported for EAEC-posi-tive individuals support the hypothesis of a genetic predispositionfor the manifestation of illness. Other host factors that may beimportant for EAEC pathogenesis require further investigation.
Immunological Response to EAEC Infection
Dispersin has been shown to cause an immune response in trav-elers visiting Mexico. Five students with diarrhea were diagnosedas having EAEC in their stool samples, and they showed a signifi-cant increase in anti-dispersin IgG absorbance when serum sam-ples were compared before and after infection measured by anenzyme-linked immunosorbent assay (ELISA) (P � 0.01). Stu-dents with and those without diarrhea showed significantly higherlevels of IgG antibody to a recombinant dispersin protein uponreturning from Mexico than the levels measured in serum at base-line (P � 0.0001). However, students who tested positive for dis-persin antibodies at baseline were not protected from EAEC in-fection during their stay (85). In addition, dispersin has not beenfound to be restricted to EAEC strains, as it has also been detectedin nonpathogenic E. coli strains (112). Another study showed in-creased levels of IgA antibodies binding to crude EAEC antigens instool samples from 5 out of 10 American students returning fromGuadalajara, Mexico. In that study, EAEC strain JM221 was usedas the antigen in dot blot and Western blot analyses (113). A vol-unteer study also showed increased levels of antibodies to differentantigen preparations from EAEC strain JM221 measured in stoolsamples from patients who developed diarrhea after oral challengewith strain JM221. Five patients who did not develop diarrhea inthe trial period proved to have increased secretory IgA (sIgA) an-tibody levels in stool samples before challenge, suggesting possibleantibody protection (114). In a Brazilian case-control study, in-creased serum levels of IgM and IgG antibodies to the toxins Petand Pic were measured in children following an EAEC infection;however, antibodies to Pic were also observed in healthy children.No obvious correlation between EAEC strains carrying the pic andpet genes isolated from children and measured antibodies to Picand Pet were seen (77). The conclusion from these studies is that
none of the antibodies measured have proven to offer high speci-ficity for EAEC, which excludes their applicability in a clinicalsetting.
MULTIDRUG RESISTANCE IN EAEC STRAINS
A worrying high level of multidrug resistance among EAEC strainshas been reported in several studies (8, 52, 115, 116). In addition,extended-spectrum beta-lactamase (ESBL) production and in-creased resistance to quinolones in EAEC strains have been de-scribed (108, 115–117). A study conducted in southern India(118) investigated 64 EAEC strains and reported that 75% of thestrains were multidrug resistant, and resistance to ciprofloxacinwas found in 63.5% of the strains. The majority of the multidrug-resistant strains were identified in children �5 years old; this maybe caused by the higher prevalence of EAEC in this age group andby colonization rather than infection, leading to a long-term car-rier state, which may facilitate antibiotic pressure. This statementis supported by another study investigating ESBL production byEAEC strains, which was detected in 1.5% of children with diar-rhea and in 4.3% of children without diarrhea (determined byPCR targeting the CTX-M enzyme) (108). This study was per-formed in 2008 to 2009 in Nicaragua, and EAEC strains werefound to have levels of resistance to ampicillin and trimethoprim-sulfamethoxazole that were significantly higher than those ofEHEC and EPEC strains (P � 0.005) and to have higher levels ofresistance to amoxicillin than ETEC strains (P � 0.021).
In an Iranian study, multidrug resistance in 10 EAEC strainswas investigated, and it was found in 71.4% of strains, with re-duced resistance to ciprofloxacin being detected in 42.9% ofstrains (52); however, the small number of strains tested in thisstudy does not allow a generalized picture of resistance of EAECstrains. Another study investigated the presence of ESBL-produc-ing strains and detected ESBL in 5 of 51 EAEC strains isolatedfrom Spanish travelers who had visited India (116). The GermanEAEC O104:H4 outbreak strains showed ESBL production (99),and of Danish EAEC UTI O78:H10 outbreak strains, 37% werefound to be multidrug resistant (81). The conclusion from thestudies mentioned above is that although the proportion of ESBL-producing EAEC strains is small, it is still a cause for concern, andsuch strains are being increasingly reported. In the battle againstthe development of further antibiotic resistance, the use of fluidreplacement and supportive treatment is preferable for manage-ment of EAEC-induced diarrhea in uncomplicated cases. Empir-ical antimicrobial therapy of EAEC should be avoided when anti-biotic intervention is deemed necessary.
RESERVOIR AND TRANSMISSION OF EAEC
The reservoir for EAEC still has not been determined, but it isgenerally accepted to be human (97, 115, 119). The transmissionof EAEC is often described as being food borne or through con-taminated water, and as such, it is believed to be transmitted by thefecal-oral route (11). One study investigated the growth of EAECin drinking water (120), where bacteria were added to differentsources of bottled water. It was discovered that EAEC strainsmaintained their viability for up to 60 days at normal storagetemperatures. The survival of the bacteria was discovered to beprolonged in mineral water compared to spring water. This wassuggested to be due to the larger amounts of Ca2� and Mg2� inmineral water, inducing genetic competence. Surface water,which in some regions of Australia is a source of drinking water,
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was investigated for the presence of pathogenic E. coli from vari-ous creeks, rivers, and drains before and after a storm in Brisbane,Australia. EAEC was identified by PCR targeting the aggR gene in36% of the water samples in the dry period and in 26% in the wetperiod and was found to be highly prevalent among the diarrhea-genic E. coli strains investigated (121). In a different study, watersamples from 46 aquatic locations in Bangladesh were collectedduring both the winter and summer seasons, and those investiga-tors found EAEC by PCR targeting a primer sequence comple-mentary to the CVD432 probe in 17% of the samples in the winterseason and 4% in the summer season (122). Another possibleroute of transmission of EAEC is food handling, which was inves-tigated in a study performed in Ouagadougou, Burkina Faso,where EAEC was detected by PCR targeting the pic and aggRgenes. EAEC was detected in 5 samples out of 120 samples ofdifferent meats and beef (123). Weaning foods as a possible routeof transmission of gastrointestinal pathogens was investigated in astudy performed in São Paolo, Brazil, and EAEC was found byDNA hybridization in 3 out of 100 milk samples from infant feed-ing bottles that were handled by mothers with low socioeconomicstatus (124). Animals as possible reservoirs of EAEC were investi-gated in a British study (125), and 1,227 E. coli isolates were inves-tigated as possible EAEC strains: 401 from cows, 406 from sheep,and 400 from pigs. However, no EAEC isolates were detected byusing the pAA probe. A different study investigated diarrheagenicE. coli strains from cats and dogs in Brazil and detected EAEC byPCR targeting the aggR gene. EAEC was detected in 7.4% of dogswith diarrhea, 3.9% of dogs without diarrhea, and one cat withoutdiarrhea (126). A French study investigated whether slaughter-house waste is a possible source of EAEC in rivers, but EAEC wasnot detected in wastewater or effluents by PCR targeting the aggR,aap, and aatA genes (127). EAEC has been isolated only rarelyfrom animal sources, and whether animals truly are potential res-ervoirs of EAEC is not conclusive. Whether companion animalsare accidental hosts of EAEC due to close contact with humans canonly be speculated upon. Sharing of E. coli strains among house-hold members and pets has been reported previously (128). Theabove-mentioned studies strongly indicate that one likely route oftransmission of EAEC is via contaminated food and water, butfurther research to exclude nonhuman reservoirs of EAEC shouldbe conducted.
EAEC AS A CAUSE OF CHILDHOOD DIARRHEA INDEVELOPING COUNTRIES
Early studies on the etiology of diarrhea in children living in de-veloping countries revealed that EAEC is highly prevalent (10, 93,129). A case-control study from 2004 (67) investigated the causeof diarrhea in Mongolian children. Here, EAEC was the most fre-quently detected type of DEC. Only EAEC strains with the aggRgene were found to be associated with diarrhea. A possible con-founder in the study may have been gastrointestinal viruses, forwhich no testing was performed. In 1999, a case-control study wasperformed in Kolkata, India, where childhood diarrhea was inves-tigated, and EAEC strains were detected in stool samples by aHeLa cell assay and PCR amplification of a gene product comple-mentary to the CVD432 probe. EAEC was found to be associatedwith diarrhea in children �36 months of age (130). In this study,watery diarrhea was reported more frequently than mucoid diar-rhea among EAEC-positive children (72% versus 28%). A studyby Steiner et al. in 1998 (93) found that growth impairment and
increased levels of fecal inflammation markers were associatedwith EAEC infection in children living in a Brazilian urban slum.EAEC was detected by the AA gene probe and the HEp-2 cell assay,and EAEC-positive children in this study had impaired growthregardless of clinical manifestations (Table 3). Another case-con-trol study investigating childhood diarrhea failed to detect anyassociation with EAEC, but there was a high rate of carriage ofEAEC among controls. This study was performed in Dhaka, Ban-gladesh, from 1993 to 1994, and EAEC was detected by DNAhybridization using the CVD432 probe (131, 132). A high rate ofcarriage of EAEC in young children has been reported by severalother studies. EAEC was found in greater numbers in the controlgroup in a case-control study (133) conducted between 2003 and2006 in South India. This study investigated the etiology of diar-rhea in children �5 years of age. EAEC was detected by multiplexPCR targeting the astA, aap, aggR, and aafII genes and by PCRamplification of a product corresponding to the AA probe. EAECwas found in 14.7% of cases and 23.7% of controls. However,EAEC strains carrying only the aap, aggR, and genes correspond-ing to the AA probe were significantly associated with diarrhea,with incidences of 6.1% in cases and 3.0% in controls (P � 0.001).Another recent study found a high rate of asymptomatic carriersof EAEC in Mali, where diarrhea in children aged 0 to 59 monthswas investigated. After 1 year of surveillance, EAEC was found tobe the only DEC in 60 children with diarrhea and in 61 childrenwithout diarrhea. However, EAEC strains with the sepA gene werefound to be associated with diarrhea (odds ratio [OR], 5.6; P �0.0006) (42). In conclusion, the many studies on childhood diar-rhea and EAEC indicate that there is a valid association, but anexhaustive diagnostic method to detect pathogenic EAEC strainsis required, and the study design should include simultaneoustesting for conventional gastrointestinal pathogens. In addition,to assess the pathogenicity of EAEC in childhood diarrhea in gen-eral, case-control studies should be conducted in a Western set-ting to minimize or eliminate contributing disease-related factorssuch as malnutrition and poor hygiene.
ASSOCIATION BETWEEN EAEC AND HIV-RELATED DIARRHEA
HIV-related diarrhea was linked to EAEC in early studies, andEAEC was considered an opportunistic pathogen. A Swiss studyconducted between 1996 and 1998 investigated HIV-related diar-rhea (134), where EAEC was detected by the HeLa cell assay, theCVD432 probe, and PCR primers complementary to the CVD432probe. In this study, a low CD4 lymphocyte count was not foundto be a predisposing factor for EAEC infection. Of the 7 patientswho experienced diarrhea, 4 later suffered from persistent diar-rhea or chronic intermittent diarrhea. Tests for enteric parasites,cytomegalovirus, and enteropathogenic bacteria were included inthe testing of the stools. A study conducted in Senegal (135) foundEAEC in 31 of 158 HIV patients with diarrhea and in 3 of 160 HIVpatients without diarrhea. EAEC was detected by PCR amplifica-tion of a product corresponding to the CVD432 probe (136). Inthat study, EAEC was defined as an opportunistic pathogen. Anassociation between EAEC and diarrhea has not been reported byall studies on HIV-related diarrhea. EAEC was found in equalnumbers in diarrhea cases and controls among HIV-positive chil-dren in Peru (137), where EAEC was detected by PCR targetingthe aggR gene. Another study found that HIV patients were morelikely to be EAEC positive: 29.5% versus 14% in the HIV-negativegroup. EAEC was detected by multiplex PCR targeting aggR and
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aap, the AA probe, and quantitative real-time PCR targeting theaggR gene (94). EAEC was found to be associated with occultblood in stool samples, (OR, 5.096; confidence interval, 2.665 to9.644), and an association between bacterial load and the mani-festation of illness was reported.
EAEC continues to play an important role in detection of thecause of diarrhea in HIV patients. However, the lack of an associ-ation between diarrhea in EAEC-positive patients and CD4 lym-phocyte counts does not support the theory of opportunisticpathogenicity, and today, the association between EAEC and di-arrhea is not limited to this group of patients.
TRAVELER’S DIARRHEA AND EAEC
More recent studies have found an association between EAEC andtraveler’s diarrhea. Initially, this was found for patients returningfrom developing countries, but now, the association has been re-ported in cases of traveler’s diarrhea obtained from various desti-nations. One study of traveler’s diarrhea concluded that the EAECvirulence factors aggR and aggA were the most prevalent EAECgenes detected in European and American patients with diarrheareturning from Montego Bay, Jamaica; Goa, India; and Guadala-jara, Mexico (11). Another study found the combination of theaggR gene and the aat gene to be associated with traveler’s diar-rhea; this combination was detected in 45% of cases, as opposed to16.4% of controls (P � 0.01). One strength of this study was thatcases of traveler’s diarrhea were compared with travelers who didnot have diarrhea and who were returning from the same regionsin the tropics of Asia, Africa, and Latin America (138). An Amer-ican study (29) investigated cases of traveler’s diarrhea from India,Guatemala, and Mexico. Forty-nine travelers had diarrhea andEAEC, 15 had EAEC without diarrhea, and 17 neither had diar-rhea nor tested positive for EAEC. EAEC strains with the aggR,astA, set1A, and aap genes were associated with traveler’s diarrhea(P � 0.05). Coinfections were reported to occur at a high fre-quency in another study of patients returning from India, Ja-maica, and Mexico (139). The most frequent pathogen isolated inthat study was ETEC, which was found in 41 out of 72 mixedinfections with EAEC detected by the HEp-2 cell assay. The pres-ence of another well-established pathogen, i.e., coinfection, inthese patients’ stool samples complicates any association betweendiarrhea and EAEC. A high number of coinfections was reportedby a study investigating traveler’s diarrhea among patients whohad visited Benin, West Africa (140). This study found that 79% ofthe patients were coinfected, where EPEC and EAEC were themost frequently detected pathogens. Coinfections with EAECwere also mentioned in another study but were described only asbeing scattered among cases of traveler’s diarrhea. This study in-vestigated Korean patients returning from Southeast Asian coun-tries (141), where ETEC had the highest incidence of entericpathogens detected (found in 36.0% of cases), followed by EAEC(found in 27.0% of cases). EAEC was detected by PCR, but theprimers used in this study were not described. Other pathogensdetected were Vibrio parahaemolyticus, Vibrio cholerae, Salmonellaspecies, Shigella, and norovirus.
EAEC is often isolated in cases of traveler’s diarrhea from dif-ferent countries, but the strong association found may have beenoverestimated due to the vast number of studies on EAEC per-formed for this patient category. The large number of coinfectionsand the lack of exhaustive testing for enteric pathogens in thesepatients mean that the general assessment of the pathogenic con-
tribution of EAEC to cases of traveler’s diarrhea must still be re-garded as questionable.
ASSOCIATION BETWEEN EAEC AND DIARRHEA ININDUSTRIALIZED COUNTRIES
In studies of the etiology of diarrheal episodes in industrializedcountries, the prevalence of EAEC has been reported to be high. Astudy conducted in Germany in 1997 found EAEC to be the thirdmost commonly isolated microorganism in young children withdiarrhea (2%), after Salmonella species (13.4%) and STEC (3.1%).EAEC infection was diagnosed in 16 cases by using the HEp-2 cellassay and PCR primers complementary to the CVD432 probe.EAEC was not detected in an asymptomatic group of children(119). However, a prospective American study did not find anassociation between EAEC and childhood diarrhea in the commu-nity setting (142). This study involved 604 healthy 6- to 36-month-old children who were monitored over a 6-month period,and the incidence of diarrhea was reported to be 2.2 per person-year. Children with intestinal malabsorption, inflammatory boweldisease, or cystic fibrosis and those who were treated with antibi-otics were excluded. Testing for enteric viruses, parasites, and en-teropathogenic bacteria was performed. Whether or not the ex-clusion of children with the comorbidities mentioned above inthis study played an important part in the pathogenic potential ofEAEC can only be speculated upon but warrants further research.An investigation of possible host factors contributing to EAEC-induced disease in children in industrialized countries should beconducted. A case-control study performed in Cincinnati, OH,found that children �1 year of age in the case group had EAECisolated significantly more frequently from stool samples than didchildren in the control group. Interestingly, pathogenic EAECstrains were identified only by the CVD432 probe and not by theaggregative adherence pattern observed in the HEp-2 cell assay.Strains with the aggregative adherence phenotype were equallydistributed between groups (143). Aggregative adherence in theHEp-2 cell assay was observed in E. coli strains that did not possessany known EAEC genes in a British study investigating commu-nity-acquired diarrhea (73). The HEp-2 cell assay was reported todetect various EAEC genotypes. This study used PCR primers tar-geting the aat, aaiA, and astA genes. Stool samples were collectedas part of a routine examination for diarrhea by the patients’ gen-eral practitioner, which included testing for EPEC, Salmonellaspecies, Shigella sonnei, Shigella flexneri, Clostridium difficile, Cam-pylobacter species, enteric parasites, and viruses. EAEC strainswith a combination of the aat, aai, and/or astA gene were found in39 of 500 patients with diarrhea. Some EAEC-positive patientswere coinfected with, for example, Campylobacter species (3 cases)and rotavirus (1 case). Foreign travel was associated with only aminor proportion of the EAEC-positive cases.
EAEC was associated with diarrhea in a national surveillancestudy conducted from 1993 to 1996 and from 2008 to 2009 in theUnited Kingdom (144). It was shown that approximately 3.3% ofintestinal infectious diseases were associated with EAEC, and theburden of EAEC was measured by quantitative PCR amplificationof a product corresponding to the CVD432 probe. However, theEAEC burden determined by quantitative PCR was not useful fordiscrimination between EAEC strains isolated from positive casesand EAEC strains isolated from controls.
In conclusion, EAEC is frequently isolated in industrializedcountries, not only from outbreaks, and should be considered
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for inclusion in a diagnostic algorithm for enteric pathogens inthese settings. However, more case-control studies should beperformed in Western countries to identify the possible patho-genic contribution of EAEC to diarrheal episodes in theseareas.
URINARY TRACT INFECTIONS AND EAEC
Recently, EAEC strains have been associated with urinary tractinfections (UTIs) (46, 100, 101, 145). An outbreak of UTI casesassociated with an EAEC strain also possessing extraintestinalpathogenic E. coli (ExPEC) genes was reported in 1991 in a Danishstudy (81). In that outbreak, 18 patients suffering from UTI in theCopenhagen area were found to be infected by strains of the sameserotype, O78:H10, and the same phylogenetic group (group A).The outbreak strain had the ExPEC genes fyuA, traT, and iutA andthe EAEC genes sat, pic, aap, aaiC, aatA, aggR, and aggA. In a laterstudy, which further characterized the Copenhagen outbreakstrains, it was shown that most of the 19 strains were capable ofbiofilm formation. One of the outbreak isolates, originating froma child aged 4 months with recurrent UTI, proved to form exten-sive biofilms and pronounced adhesion to human bladder epithe-lial cells by the expression of AAF/I (34). This indicates thatEAEC-associated virulence factors enhance uropathogenicity, andthe possession of these virulence factors may have promoted thecapability of the strain to cause the outbreak.
A case of urinary tract infection preceded by diarrhea caused byEAEC was reported in a 55-year-old immunocompromised fe-male, leading to bacteremia and urosepsis (102). In this study, anEAEC strain with the same pulsed-field gel electrophoresis(PFGE) profile was isolated from stool, urine, and blood culturesamples from this patient. The strain was serotyped as O176:NTand tested positive for the CVD432 probe and the aggR, aap,agg3C, astA, pic, aaiC, and air genes by PCR. In a Korean study(145), the cause of UTI in small children was investigated by ana-
lyzing suprapubic urine specimens from children with and thosewithout UTI (as defined by numbers of CFU in urine). EAEC wasdetected by PCR targeting the aggR and aap genes in children withcases of UTIs; however, strains with the combination of aggR andaap were found in significantly higher numbers in the controlgroup (P � 0.03). EAEC isolated from patients with UTI, withdifferent combinations of the EAEC genes aap, aggR, aggC, astA,agg3C, pet, and pic but with all strains containing genes encodingAAF/I, were reported in a Brazilian study (100). These authorssuggested that this fimbria might play an important role in theuropathogenesis of EAEC.
In general, characterization of E. coli isolated from UTI cases islimited in the clinical setting, which obscures the pathogenic con-tribution from different uropathogenic E. coli groups. However,true outbreaks of UTI cases are rare, implicating little knowledgeon this subject. Clearly, the role of EAEC in uropathogenesisshould be further investigated.
OUTBREAKS
Outbreaks of gastroenteritis linked to EAEC have been reported(Fig. 3). One outbreak took place in a Serbian nursery (80) in1995, where EAEC was detected by the HEp-2 cell assay and wascharacterized as belonging to serotype O4 in 12 of 19 babies whohad low-grade fever, diarrhea, and weight loss. Rotavirus was theonly gastrointestinal virus screened for. PCR was not performedon the outbreak strain. Another outbreak took place in a policeinstitute in Japan in 2005, where the staff experienced gastroen-teritis after having consumed food suspected of being contami-nated with EAEC (79). Four staff members and one food handlertested positive for EAEC in stool samples; the isolates were foundto have an identical serotype (O126:H27). However, no microbi-ological investigations for enteric viruses were done for this out-break. The largest reported outbreak of EAEC, apart from the2011 German O104:H4 EAEC outbreak, took place in the Gifu
FIG 3 Geographic distribution of EAEC outbreaks. N is the number of patients with confirmed EAEC involved in the outbreak. The total numbers of patientsinvolved in outbreaks of diarrhea are given in brackets. The Danish outbreak was an outbreak of EAEC causing urinary tract infections. References are shown inparentheses. HUS, hemolytic-uremic syndrome.
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prefecture, Tajimi, Japan, in 1993 (90). Here, 2,697 children de-veloped signs of food poisoning after having consumed schoollunches. Fecal specimens were collected from 30 children withsevere protracted diarrhea, and 12 tested positive for EAEC by theHEp-2 cell assay and the astA gene by PCR; the strains were all Ountypeable:H10. However, the children in this outbreak were nottested for gastrointestinal viruses, to which the symptoms corre-spond, including short-term gastroenteritis, nausea, stomachache, and diarrhea, which affected a considerable number of thecases in the outbreak. Another EAEC outbreak took place in 2008in Italy at a farm holiday resort, where the guests experiencedgastroenteritis after having consumed unpasteurized cheese (78).EAEC was isolated in stool samples from 6 restaurant guests and 1staff member; the strains were identified by the HEp-2 cell assay.The EAEC strain involved was identified as belonging to serotypeO92:H33, and it tested positive for the virulence genes aggR, aat,aap, and set1A by PCR. Testing for norovirus, but not rotavirus,was performed in that study.
The outbreaks mentioned above show a considerable potentialfor food-borne transmission of EAEC. The lack of internationalsurveillance of EAEC could lead to missed outbreaks caused byEAEC.
The German O104:H4 EAEC Outbreak in 2011
In May 2011, a large food-borne outbreak of Shiga toxin-produc-ing E. coli (STEC) occurred in Germany, with 3,842 confirmedcases. The source of the food-borne outbreak was bean sprouts,which caused 855 (22%) cases of hemolytic-uremic syndrome andconsiderable mortality (amounting to 54 [1.4%] deaths) (146).The outbreak strain was found to show the characteristic stacked-brick pattern when grown on HEp-2 cells, and it tested positive forthe EAEC genes aggR, aggA, set1, pic, and aap (99). The outbreakstrain was serotyped as O104:H4 and contained a prophage en-coding the Shiga toxin 2 gene, stx2, which is normally absent inEAEC strains (98). Other, considerably smaller, occurrences ofEAEC-STEC hybrid stains have been reported (147–149). An out-break of HUS in 10 children in France in 1992 was associated withan EAEC strain of serotype O111:H2, which was positive for thestx2 gene (150). A 3-year-old boy from Japan was diagnosed withHUS in 1999, where an EAEC strain of serotype O86:HNM whichwas positive for the stx2 gene was identified (148). An EAEC O111:H21 strain with the stx2 gene was found to be associated with anoutbreak of HUS in a household in Ireland in 2012 (149). AnEAEC strain of serotype O104:H4, but lacking the Shiga toxin, waspreviously detected in an HIV-positive patient in the Central Af-rican Republic who was suffering from persistent diarrhea (22).Further characterization of the outbreak strain by whole-genomephylogenetic analysis showed a cluster of genes with a close geneticrelationship between the African non-Shiga toxin-positiveO104:H4 strain and the outbreak strain (98), which suggested arecent acquisition of the phage carrying stx2. The outbreaks men-tioned above show the considerable potential of EAEC to acquireadditional virulence through genetic exchange, and this furtheremphasizes the need for microbiological surveillance.
DISCUSSION
The CVD432 probe targeting the aat gene is widely used for thedetection of EAEC by DNA hybridization (17, 52, 69, 79, 119,131). However, this probe fails to detect all EAEC strains showingthe stacked-brick pattern by the HEp-2 cell assay (65, 69, 106). The
possibility of interobserver variability in pattern recognition wasmentioned in a study by Pabst et al. (151), which could lead todifferent rates of detection of EAEC between studies. Indeed,strain JM221 was first described to show localized adherence (64)but has since been said to show aggregative adherence (63). Onecase-control study found patterns of adherence of EAEC strains(found by PCR testing for the aatA and aggR genes) to be diverse,including nonadherent, sparsely adherent, and densely adherentpatterns, without any correlation with cases or controls (95).
PCR is a less demanding and more objective method for thedetection of pathogenic E. coli. Extensive studies have been per-formed by using PCR to find the right combination of genes iden-tifying the “truly pathogenic” EAEC strains (51, 65, 69, 79, 105,152, 153); however, no consensus has been reached on this matter.As suggested by another study (20), the combination of EAECgenes might depend on the geographic region, and recognitionof these various combinations of genes could lead to a betterunderstanding of their role in the pathogenicity of specificEAEC strains.
The aggR gene is highly conserved among EAEC strains and hasbeen found to be associated with diarrhea in several studies (105,133, 154). A strong correlation between the CVD432 probe andthe aggR gene has been reported (7, 52, 69). Quantitative PCRtargeting the aggR gene has shown dose dependency (94) in themanifestation of illness. However, several other studies have notbeen able to associate aggR with disease (42, 65, 74, 155). Anotherway of quantifying the EAEC burden was used in a study usingDNA extraction from stools, which showed a log-linear correla-tion between CFU of E. coli per gram of stool and median fluores-cence intensity determined by multiplex PCR-Luminex technol-ogy. A correlation between the aatA gene and CFU of EAEC instool was found. Further research on this method, combined withclinical information, could reveal a possible correlation betweenthe degree of illness and the EAEC burden (156).
Persistent diarrhea has frequently been mentioned in thestudies reviewed, but it appears to lack solid documentation.Several studies have found a low prevalence of persistent diar-rhea in EAEC-positive patients (86, 129, 151, 157), which callsinto question the importance of this disease trait in an EAECinfection in general. Despite the genetic heterogeneity of EAECstrains, the reported clinical manifestations have a high level ofhomogeneity (8, 86, 134, 158, 159), with symptoms such aslow-grade fever, watery or mucoid diarrhea, borborygmi, andloss of appetite.
In a number of studies, EAEC has been isolated in almostequal numbers from patients suffering from diarrhea and fromcontrol groups (15, 133, 137). This finding has been proposedto be a result of acquired immunity (105, 137) due to poorsanitary conditions as well as frequent exposure to pathogenicE. coli strains, resulting in the development of protective antibod-ies and colonization rather than infection. This has been stated instudies conducted in developing countries. However, high num-bers of asymptomatic carriers of EAEC have also been observed incountries such as the United States, Switzerland, and Denmark(40, 72, 109, 142, 143). Case-control studies performed in settingswith poor hygiene are difficult to interpret in terms of key EAECvirulence genes, due to the high prevalence of EAEC in these areas.The reported long-term carrier state and the low pathogenicity ofEAEC might lead to colonization rather than infection, whichcould lead to incorrect conclusions about truly pathogenic EAEC
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virulence genes when EAEC strains are compared between casesand controls. As was mentioned previously (95), the case-controlstudy design performed in a community with a high carrier rate ofEAEC complicates the assessment of pathogenicity.
Coinfections, especially in traveler’s diarrhea, are said to occurfrequently (138); however, few studies have investigated the etiol-ogy of diarrhea, including testing for DEC, viruses, and parasites,thus leaving the pathogenicity of EAEC difficult to assess in thesecases. In addition, possible synergy between these microorgan-isms should be investigated.
CONCLUSIONS
EAEC has been isolated from many patients suffering from diar-rhea in a number of regions of the world and from various socio-economic strata. It is most frequently reported as a self-limitingdiarrheagenic pathogen that generally results in mild symptoms.The importance of host factors as determinants of EAEC patho-genesis should be investigated in depth. Persistent diarrhea causedby EAEC is often mentioned in studies, but further research onthis matter is needed. The genetic heterogeneity of EAEC compli-cates international surveillance and therapeutic approaches con-siderably. The lack of a specific diagnostic algorithm to detectEAEC makes our understanding of the pathogenicity of EAECespecially difficult.
ACKNOWLEDGMENTS
We thank Rie Jønsson, Department of Microbiology and Infection Con-trol, Statens Serum Institut, for inspiring discussions and Fig. 1. We alsothank Marian Jørgensen, Department of Microbiological Diagnostics andVirology, Statens Serum Institut, for critical comments. We are grateful toAnja Bjarnum for assistance with graphical illustrations.
Funding was provided by Danish Council for Strategic Research, In-novation and Higher Education grant number 2101-07-0023 to KarenAngeliki Krogfelt.
We declare that there are no conflicts of interest.
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Carsten Struve, M.Sc., Ph.D., is a Senior Scien-tist at the Department of Microbiology and In-fection Control, Statens Serum Institut, Den-mark. He did his M.Sc. studies at the Universityof Copenhagen and completed his Ph.D. in mo-lecular microbiology in 2002. His research fo-cuses on the identification and characterizationof virulence and resistance mechanisms in En-terobacteriaceae, including the developmentand use of experimental infection models.
Katharina E. P. Olsen, Ph.D., Pharm.D., is theHead of the National Reference Laboratory forEnteropathogenic Bacteria at the Departmentof Microbiology and Infection Control, StatensSerum Institut (SSI), Copenhagen, Denmark,and is responsible for the national surveillanceof Clostridium difficile. Formerly, she held aone-year position as a researcher at the WHOInternational Collaborating Centre for Refer-ence and Research on Escherichia and Klebsiella,SSI. She was educated at the School of Pharma-ceutical Sciences, Faculty of Health and Medical Sciences, Copenhagen Uni-versity, where she also earned her Ph.D. in medical molecular biology. Dr.Olsen is an officially appointed examiner in Microbiology at CopenhagenUniversity, Faculty of Health and Medical Sciences. She is a member of theDanish Society for Clinical Microbiology, the Danish National Committeeunder the Nordic Committee on Food Analysis, and the ESCMID StudyGroup for Clostridium difficile (ESGCD). Her research focuses on molecularepidemiology, diagnostics, and cytotoxicity of bacterial protein toxins.
Betina Hebbelstrup Jensen qualified in medi-cine at Aarhus University, Denmark, and Co-penhagen University, Denmark, in 2010. Part ofher medical internship was conducted at theDepartment of Gastroenterology, GlostrupHospital, Copenhagen, Denmark. Currently,she is performing her Ph.D. studies at StatensSerum Institut, Department of Microbiologyand Infection Control, on enteroaggregativeEscherichia coli, with a focus on the clinical im-plications associated with this microorganism.
Andreas Munk Petersen completed his M.D.and Ph.D. in Gastroenterology at the Universityof Copenhagen. He completed a residency ininternal medicine and gastroenterology at Her-lev University Hospital, Denmark, before con-tinuing his employment as a consultant at Hvi-dovre University Hospital, Denmark. Hisresearch focuses on the interfaces between mi-crobiology and gastroenterology.
Karen Angeliki Krogfelt, M.Sc. (Eng.), Ph.D.,is an adjunct professor at the Institute of Sys-tems Biology, Technical University of Den-mark, DTU, and Head of a research unit at theDepartment of Microbiology, Statens SerumInstitut. She is involved in teaching master’scourses and has supervised numerous master’sand Ph.D. students. Her main research activi-ties focus on the pathogenic mechanisms re-lated mainly to intestinal bacterial infectionsand on assessing the development of gut micro-biota and its relation to pathogenesis. Characterization of specific virulencefactors involved in pathogenesis is also used for the development of diagnos-tic methods for bacterial infections.
Hebbelstrup Jensen et al.
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