Ecoli PDF

CLINICAL MICROBIOLOGY REVIEWS, Jan. 1998, p. 142–201 Vol. 11, No.
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0893-8512/98/$04.0010
Copyright © 1998, American Society for Microbiology
Diarrheagenic Escherichia coli

JAMES P. NATARO* AND JAMES B. KAPER
Center for Vaccine Development, Departments of Medicine, Pediatrics, and Microbiology & Immunology,
University of Maryland School of Medicine, Baltimore, Maryland 21201
INTRODUCTION .......................................................................................................................................................144
ISOLATION AND IDENTIFICATION....................................................................................................................144
Biochemicals ............................................................................................................................................................144
Serotyping ................................................................................................................................................................144
Phenotypic Assays Based on Virulence Characteristics ....................................................................................144
Molecular Detection Methods ...............................................................................................................................145
Nucleic acid probes.............................................................................................................................................145
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PCR.......................................................................................................................................................................147
COMMON THEMES IN E. COLI VIRULENCE...................................................................................................147
ENTEROTOXIGENIC E. COLI ...............................................................................................................................147
Pathogenesis ............................................................................................................................................................148
Heat-labile toxins ................................................................................................................................................148
(i) LT-I .............................................................................................................................................................148
(ii) LT-II...........................................................................................................................................................149
Heat-stable toxins ...............................................................................................................................................149
(i) STa ..............................................................................................................................................................149
(ii) STb .............................................................................................................................................................151
Colonization factors............................................................................................................................................151
Epidemiology ...........................................................................................................................................................152
Clinical Considerations..........................................................................................................................................153
Detection and Diagnosis ........................................................................................................................................154
ENTEROPATHOGENIC E. COLI............................................................................................................................155
Pathogenesis ............................................................................................................................................................155
Attaching-and-effacing histopathology .............................................................................................................155
Three-stage model of EPEC pathogenesis.......................................................................................................156
(i) Localized adherence..................................................................................................................................156
(ii) Signal transduction..................................................................................................................................156
(iii) Intimate adherence .................................................................................................................................158
Secreted proteins.................................................................................................................................................158
Locus of enterocyte effacement .........................................................................................................................159
EAF plasmids ......................................................................................................................................................159
Regulation ............................................................................................................................................................160
Other potential virulence factors......................................................................................................................160
(i) Other fimbriae ...........................................................................................................................................160
(ii) EAST1 ........................................................................................................................................................160
(iii) Invasion ....................................................................................................................................................160
Mechanism of diarrhea......................................................................................................................................161
Epidemiology ...........................................................................................................................................................161
Age distribution...................................................................................................................................................161
Transmission and reservoirs.............................................................................................................................161
EPEC in developed countries............................................................................................................................161
EPEC in developing countries ..........................................................................................................................162
Definition of EPEC .............................................................................................................................................162
Diagnostic tests ...................................................................................................................................................163
(i) Phenotypic tests.........................................................................................................................................163
(ii) Genotypic tests .........................................................................................................................................163
* Corresponding author. Mailing address: Center for Vaccine De-

velopment, Departments of Medicine and Pediatrics, University of
Maryland School of Medicine, Baltimore, MD 21201. Phone: (410)
706-8442. Fax: (410) 706-6205. E-mail: jnataro@umaryland.edu.
142
VOL. 11, 1998 DIARRHEAGENIC E. COLI 143
ENTEROHEMORRHAGIC E. COLI .......................................................................................................................164

Origins......................................................................................................................................................................164
Pathogenesis ............................................................................................................................................................165
Histopathology.....................................................................................................................................................165
Shiga toxins .........................................................................................................................................................165
(i) Structure and genetics..............................................................................................................................165
(ii) Stx in intestinal disease ..........................................................................................................................165
(iii) Stx in HUS...............................................................................................................................................167
EAST1...................................................................................................................................................................167
Enterohemolysin..................................................................................................................................................167
Intestinal adherence factors ..............................................................................................................................167
pO157 plasmid ....................................................................................................................................................168
Iron transport......................................................................................................................................................168
Other potential virulence factors......................................................................................................................168
Epidemiology ...........................................................................................................................................................169
Incidence ..............................................................................................................................................................169
Animal reservoir .................................................................................................................................................169

Transmission .......................................................................................................................................................169
Non-O157:H7 serotypes......................................................................................................................................170
Clinical disease ...................................................................................................................................................171
Treatment.............................................................................................................................................................171
Vaccines................................................................................................................................................................172
Diagnosis and Detection ........................................................................................................................................172
General considerations ......................................................................................................................................172
(i) Why and when to culture .........................................................................................................................172
(ii) Biosafety issues.........................................................................................................................................173
(iii) Diagnostic methods.................................................................................................................................173
Culture techniques..............................................................................................................................................174
Immunoassays .....................................................................................................................................................174
(i) O and H antigens ......................................................................................................................................174
(ii) Shiga toxins...............................................................................................................................................175
(iii) Other antigens.........................................................................................................................................175
(iv) Immunomagnetic separation .................................................................................................................175
(v) Free fecal cytotoxic activity .....................................................................................................................175
DNA probes and PCR ........................................................................................................................................176
(i) Detection of stx genes................................................................................................................................176
(ii) Detection of eae genes..............................................................................................................................176
(iii) Detection of the pO157 plasmid/hemolysin gene................................................................................176
(iv) Detection of other genes .........................................................................................................................176
Strain subtyping..................................................................................................................................................177
Serodiagnosis.......................................................................................................................................................177
ENTEROAGGREGATIVE E. COLI .........................................................................................................................178
Pathogenesis ............................................................................................................................................................178
Histopathology.....................................................................................................................................................178
Adherence.............................................................................................................................................................179
EAST1...................................................................................................................................................................179
Invasiveness .........................................................................................................................................................179
Cytotoxins ............................................................................................................................................................179
Model of EAEC pathogenesis............................................................................................................................179
Epidemiology ...........................................................................................................................................................180
Clinical Features.....................................................................................................................................................181
HEp-2 assay .........................................................................................................................................................181
DNA probe ...........................................................................................................................................................181
Other tests for EAEC .........................................................................................................................................182
ENTEROINVASIVE E. COLI....................................................................................................................................182
Pathogenesis ............................................................................................................................................................182
Invasiveness .........................................................................................................................................................182
Enterotoxin production ......................................................................................................................................182
Epidemiology ...........................................................................................................................................................182
DIFFUSELY ADHERENT E. COLI .........................................................................................................................183
Pathogenesis ............................................................................................................................................................184
Epidemiology ...........................................................................................................................................................184
144 NATARO AND KAPER CLIN. MICROBIOL. REV.
Clinical Features.....................................................................................................................................................184
OTHER CATEGORIES OF E. COLI WHICH ARE POTENTIALLY DIARRHEAGENIC .............................185
CONCLUSIONS .........................................................................................................................................................185
ACKNOWLEDGMENTS ...........................................................................................................................................185
REFERENCES ............................................................................................................................................................186
INTRODUCTION methylene-blue agar, which selectively grow members of the

Enterobacteriaceae and permit differentiation of enteric organ-
Escherichia coli is the predominant facultative anaerobe of isms on the basis of morphology (32).
the human colonic flora. The organism typically colonizes the Enterobacteriaceae are usually identified via biochemical re-
infant gastrointestinal tract within hours of life, and, thereaf- actions. These tests can be performed in individual culture
ter, E. coli and the host derive mutual benefit (169). E. coli tubes or by using test “strips” which are commercially avail-
usually remains harmlessly confined to the intestinal lumen; able. Either method produces satisfactory results.
however, in the debilitated or immunosuppressed host, or For epidemiologic or clinical purposes, E. coli strains are
when gastrointestinal barriers are violated, even normal “non- often selected from agar plates after presumptive visual iden-

pathogenic” strains of E. coli can cause infection. Moreover, tification. However, this method should be used only with
even the most robust members of our species may be suscep- caution, because only about 90% of E. coli strains are lactose
tible to infection by one of several highly adapted E. coli clones positive; some diarrheagenic E. coli strains, including many of
which together have evolved the ability to cause a broad spec- the EIEC strains, are typically lactose negative. The indole
trum of human diseases. Infections due to pathogenic E. coli test, positive in 99% of E. coli strains, is the single best test for
may be limited to the mucosal surfaces or can disseminate differentiation from other members of the Enterobacteriaceae.
throughout the body. Three general clinical syndromes result
from infection with inherently pathogenic E. coli strains: (i) Serotyping
urinary tract infection, (ii) sepsis/meningitis, and (iii) enteric/
diarrheal disease. This article will review the diarrheagenic E. Serotyping of E. coli occupies a central place in the history of
coli strains, which include several emerging pathogens of these pathogens (reviewed in reference 394. Prior to the iden-
worldwide public health importance, and will specifically focus tification of specific virulence factors in diarrheagenic E. coli
on pathogens afflicting humans. We will particularly concen- strains, serotypic analysis was the predominant means by which
trate on the E. coli strains whose study has advanced most over pathogenic strains were differentiated. In 1933, Adam showed
the last decade, i.e., enteropathogenic E. coli (EPEC), entero- by serologic typing that strains of “dyspepsiekoli” could be
hemorrhagic E. coli (EHEC), and enteroaggregative E. coli implicated in outbreaks of pediatric diarrhea. In 1944, Kauff-
(EAEC). Since the categories of diarrheagenic E. coli are dif- man proposed a scheme for the serologic classification of E.
ferentiated on the basis of pathogenic features, emphasis will coli which is still used in modified form today.
be placed on the mechanisms of disease and the development According to the modified Kauffman scheme, E. coli are
of diagnostic techniques based on virulence factors. serotyped on the basis of their O (somatic), H (flagellar), and
K (capsular) surface antigen profiles (185, 394). A total of 170
different O antigens, each defining a serogroup, are recognized
ISOLATION AND IDENTIFICATION
currently. The presence of K antigens was determined origi-
Although assays to identify all categories of diarrheagenic E. nally by means of bacterial agglutination tests: an E. coli strain
coli are available, in many situations it is not necessary to that was inagglutinable by O antiserum but became aggluti-
implicate a specific E. coli pathogen in a particular patient. nable when the culture was heated was considered to have a K
Patients with enterotoxigenic E. coli (ETEC) traveler’s diar- antigen. The discovery that several different molecular struc-
rhea, for example, generally resolve their diarrhea long before tures, including fimbriae, conferred the K phenotype led ex-
they come to medical attention for stool culture. Most entero- perts to suggest restructuring the K antigen designation to
invasive E. coli (EIEC) isolates will be missed in the clinical include only acidic polysaccharides (394). Proteinaceous fim-
laboratory, yet diarrhea generally resolves and patients re- brial antigens have therefore been removed from the K series
spond to empirical antibiotics, such as fluoroquinolones, given and have been given F designations (494).
for other bacterial diarrheas. Culturing stools for most catego- A specific combination of O and H antigens defines the
ries of diarrheagenic E. coli should be performed in cases of “serotype” of an isolate. E. coli of specific serogroups can be
persistent diarrhea, especially in travelers, children and the associated reproducibly with certain clinical syndromes (Table
immunocompromised, as well as in outbreak situations. E. coli 1), but it is not in general the serologic antigens themselves
can be isolated from the stool and sent to a qualified reference that confer virulence. Rather, the serotypes and serogroups
laboratory for definitive identification. The indications for cul- serve as readily identifiable chromosomal markers that corre-
turing for EHEC differ from those for the rest of the diarrhea- late with specific virulent clones (690).
genic E. coli categories; indications for culturing EHEC are
discussed below in greater detail in the EHEC section. Phenotypic Assays Based on Virulence Characteristics
Identification of diarrheagenic E. coli strains requires that
Biochemicals these organisms be differentiated from nonpathogenic mem-
E. coli is the type species of the genus Escherichia, which bers of the normal flora. Serotypic markers correlate, some-
contains mostly motile gram-negative bacilli within the family times very closely, with specific categories of diarrheagenic E.
Enterobacteriaceae and the tribe Escherichia (55, 185). coli; however, these markers are rarely sufficient in and of
E. coli can be recovered easily from clinical specimens on themselves to reliably identify a strain as diarrheagenic. (An
general or selective media at 37°C under aerobic conditions. E. exception may be strains of serotype O157:H7, a serotype that
coli in stool are most often recovered on MacConkey or eosin serves as a marker for virulent enterohemorrhagic E. coli
TABLE 1. Serotypes characteristic of the diarrheagenic diarrheagenic E. coli is the HEp-2 adherence assay. The
E. coli categories method has recently been reviewed in detail (160). This assay
Category Serogroup Associated H antigen(s)
was first described in 1979 by Cravioto et al. (139) and remains
the “gold standard” for the diagnosis of EAEC and diffusely
ETEC O6 H16 adherent E. coli (DAEC). The HEp-2 assay has been modified
O8 H9 often since its first description, including such variations as
O11 H27 extending the incubation time to 6 h or changing the growth
O15 H11 medium during the incubation. However, collaborative studies
O20 NM
O25 H42, NM
have shown that the assay performed essentially as first de-
O27 H7 scribed provides the best ability to differentiate among all three
O78 H11, H12 adherent diarrheagenic categories (EPEC, EAEC, and
O128 H7 DAEC) (678). The HEp-2 adherence assay entails inoculating
O148 H28 the test strain onto a semiconfluent HEp-2 monolayer and
O149 H10 incubating it for 3 h at 37°C under 5% CO2. After this incu-
O159 H20 bation time, the monolayer is washed, fixed, stained, and ex-
O173 NM amined by oil immersion light microscopy. The three patterns
of HEp-2 adherence (Fig. 1), localized adherence (LA), aggre

EPEC O55 H6, NM
O86 H34, NM
gative adherence (AA), and diffuse adherence (DA), can be
O111 H2, H12, NM differentiated reliably by an experienced technician. However,
O119 H6, NM the authors have found some strains which yield equivocal
O125ac H21 results reproducibly in the HEp-2 assay.
O126 H27, NM
O127 H6, NM Molecular Detection Methods
O128 H2, H12
O142 H6 Diarrheagenic E. coli strains were among the first pathogens
for which molecular diagnostic methods were developed. In-
EHEC O26 H11, H32, NM
O55 H7
deed, molecular methods remain the most popular and most
O111ab H8, NM reliable techniques for differentiating diarrheagenic strains
O113 H21 from nonpathogenic members of the stool flora and distin-
O117 H14 guishing one category from another. Substantial progress has
O157 H7 been made both in the development of nucleic acid-based
probe technologies as well as PCR methods.
EAEC O3 H2 Nucleic acid probes. The use of DNA probes for detection
O15 H18 of heat-labile (LT) and heat-stable (ST) enterotoxins in ETEC
O44 H18 revolutionized the study of these organisms, replacing cumber-
O86 NM
O77 H18
some and costly animal models of toxin detection (455). Since
O111 H21 then, gene probes have been introduced for all diarrheagenic
O127 H2 categories. Two general methods are commonly used for nu-
O?a H10 cleic acid probe specimen preparation. The first entails the
inoculation of purified cultures onto agar plates to produce
EIEC O28ac NM “colony” blots, in which 30 to 50 such cultures are inoculated
O29 NM per plate. After incubation, the bacterial growth is transferred
O112ac NM to nitrocellulose or Whatman filter paper for hybridization
O124 H30, NM (alternatively, the cultures can be grown directly on the nitro-
O136 NM
O143 NM
cellulose overlying an agar plate). The bacterial growth on the
O144 NM paper can be lysed, denatured, and hybridized with the probe
O152 NM in situ, and then a radiographic image is generated by exposure
O159 H2, NM to X-ray film. Substantial experience by ourselves and others
O164 NM has demonstrated that the colony blot method is reliable and
O167 H4, H5, NM efficient. However, the use of this method requires that the E.
a
O antigen untypeable by conventional methods.
coli strain first be isolated from the patient’s stool, which in-
troduces the possibility that any number of E. coli colonies
picked from a stool culture may fail to yield the offending
pathogenic strain. Over several years of study, we have found
strains; nevertheless, EHEC of serotypes other than O157:H7 that patients symptomatic with E. coli diarrhea generally
are being identified with increasing frequency in sporadic and present with the pathogenic strain as their predominant E. coli
epidemic cases.) In addition to its limited sensitivity and spec- strain in the flora. Thus, studies in which three E. coli isolates
ificity, serotyping is tedious and expensive and is performed are tested per diarrheal stool specimen will have acceptable
reliably only by a small number of reference laboratories. Thus, sensitivity. If increased sensitivity is desired or if the study
detection of diarrheagenic E. coli has focused increasingly on entails a large number of asymptomatic patients, isolating five
the identification of characteristics which themselves deter- isolates per specimen may be more appropriate.
mine the virulence of these organisms. This may include in An alternative to the use of colony blots is the stool blot
vitro phenotypic assays which correlate with the presence of method. In this technique, stool samples are spotted directly
specific virulence traits or detection of the genes encoding onto nitrocellulose filters that have been overlaid onto an agar
these traits. plate (373). After overnight incubation, the filter paper is
One of the most useful phenotypic assays for the diagnosis of peeled off the plate, air dried, and treated as above for colony
146
blots. The advantages of this technique include (i) that the E. varieties. However, diarrheagenic E. coli strains possess spe-
coli colonies need not be isolated from the stool and (ii) that cific fimbrial antigens that enhance their intestinal colonizing
there may be increased sensitivity if the pathogenic strain rep- ability and allow adherence to the small bowel mucosa, a site
resents a minority member of the flora. However, the presence that is not normally colonized (389, 679). The various mor-
of large numbers of other bacteria decreases the sensitivity of phologies of E. coli fimbriae are illustrated in Fig. 2. The role
this test, and a threshold number (ca. 105 to 106 per g of stool of fimbrial structures in adherence and colonization is often
[461]) of pathogenic organisms must be present to yield defin- inferred rather than demonstrated, in part due to the host
itive results. In addition, the use of stool blots alone does not specificity of most fimbrial adhesins.
result in a pure culture of the pathogen, which may be required Once colonization is established, the pathogenetic strategies
for verification of phenotypes. of the diarrheagenic E. coli strains exhibit remarkable variety.
Nucleic acid-based probes themselves can be of two types: Three general paradigms have been described by which E. coli
oligonucleotide or polynucleotide (fragment probes). DNA may cause diarrhea; each is described in detail in the appro-
fragment (polynucleotide) probes may be derived from genes priate section below: (i) enterotoxin production (ETEC and
that encode a particular phenotype or may instead be empirical EAEC), (ii) invasion (EIEC), and/or (iii) intimate adherence
probes which, through extensive testing, are found to be linked with membrane signalling (EPEC and EHEC). However, the
with the presence of a phenotype. Although empirical probes
interaction of the organisms with the intestinal mucosa is spe-
have generated useful results (41, 701), probes which represent

cific for each category. Schematized paradigms are illustrated
the virulence genes themselves are generally superior (241).
in Fig. 3.
Oligonucleotide probes are derived from the DNA sequence
of a target gene. Annealing temperatures and other conditions The versatility of the E. coli genome is conferred mainly by
of hybridization and washing need to be determined much two genetic configurations: virulence-related plasmids and
more precisely than for polynucleotide probes. Moreover, very chromosomal pathogenicity islands. All six categories of diar-
slight strain-to-strain differences among the virulence genes rheagenic E. coli described in this review have been shown to
may generate false-negative results with oligonucleotide carry at least one virulence-related property upon a plasmid.
probes. Nevertheless, oligonucleotide probes have the advan- EIEC, EHEC, EAEC, and EPEC strains typically harbor
tage of faster and often cleaner results than those generated by highly conserved plasmid families, each encoding multiple vir-
polynucleotide methods, a factor that comes into play espe- ulence factors (275, 467, 701). McDaniel and Kaper have
cially when screening for very small genes. Recommended shown recently that the chromosomal virulence genes of EPEC
oligonucleotide probes are listed in Table 2. and EHEC are organized as a cluster referred to as a patho-
Whereas the original probe techniques involved radionucle- genicity island (431, 432). Such islands have been described for
otides to detect probe hybridization, nonisotopic methods are uropathogenic E. coli strains (163) and systemic E. coli strains
becoming more popular. These include several methods for (75) as well and may represent a common way in which the
tagging oligonucleotide probes and a smaller number of effec- genomes of pathogenic and nonpathogenic E. coli strains di-
tive techniques for detection of polynucleotide probes. These verge genetically. Plasmids and pathogenicity islands carry
nonisotopic techniques have facilitated the introduction of clusters of virulence traits, yet individual traits may be trans-
probe technology into areas where the use of radioisotopes poson encoded (such as ST) (607) or phage encoded (such as
is impractical. Shiga toxin) (485).
PCR. PCR is a major advance in molecular diagnostics of In the sections that follow, we will review all aspects of
pathogenic microorganisms, including E. coli. PCR primers disease due to the different classes of diarrheagenic E. coli.
have been developed successfully for several of the categories Since diarrheagenic E. coli strains are distinguished and de-
of diarrheagenic E. coli (listed in Table 2). Advantages of PCR fined on the basis of pathogenetic mechanisms, much of this
include great sensitivity in in situ detection of target templates. review will concern the latest advances in our knowledge of the
However, substances within stools have been shown to inter- pathogenesis of these organisms.
fere with the PCR, thus decreasing its sensitivity (615); several
methods have been used successfully to remove such inhibi-
tors, including Sepharose spin column chromatography and
ENTEROTOXIGENIC E. COLI
adsorption of nucleic acids onto glass resin (397, 615). Scru-
pulous attention to proper technique must be maintained to ETEC is defined as containing the E. coli strains that elab-
avoid carryover of PCR products from one reaction to the next. orate at least one member of two defined groups of enterotox-
ins: ST and LT (381). ETEC strains were first recognized as
COMMON THEMES IN E. COLI VIRULENCE causes of diarrheal disease in piglets, where the disease con-
Like most mucosal pathogens, E. coli can be said to follow a tinues to cause lethal infection in newborn animals (reviewed
requisite strategy of infection: (i) colonization of a mucosal in reference 15). Studies of ETEC in piglets first elucidated the
site, (ii) evasion of host defenses, (iii) multiplication, and (iv) mechanisms of disease, including the existence of two plasmid-
host damage. The most highly conserved feature of diarrhea- encoded enterotoxins. The first descriptions of ETEC in hu-
genic E. coli strains is their ability to colonize the intestinal mans reported that certain E. coli isolates from the stools of
mucosal surface despite peristalsis and competition for nutri- children with diarrhea elicited fluid secretion in ligated rab-
ents by the indigenous flora of the gut (including other E. coli bit intestinal loops (642). DuPont et al. subsequently showed
strains). The presence of surface adherence fimbriae is a prop- that ETEC strains were able to cause diarrhea in adult
erty of virtually all E. coli strains, including nonpathogenic volunteers (175).
FIG. 1. The three HEp-2 adherence patterns manifested by diarrheagenic E. coli. (A) Localized adherence (LA), typical of EPEC. Bacteria form characteristic
microcolonies on the surface of the HEp-2 cell. (B) Aggregative adherence (AA), which defines EAEC. Bacteria adhere to each other away from the cells as well as
to the cell surface in a characteristic stacked-brick configuration. (C) Diffuse adherence (DA), which defines DAEC. Bacteria are dispersed over the surface of the cell.
TABLE 2. Nucleotide sequences of PCR oligonucleotide primers and oligonucleotide probes for diarrheagenic E. coli strains
Category Factor PCR oligonucleotidesa Reference Oligonucleotide probe Reference
ETEC STI TTAATAGCACCCGGTACAAGCAGG 492 GCTGTGAATTGTGTTGTAATCC 457

CTTGACTCTTCAAAAGAGAAAATTAC GCTGTGAACTTTGTTGTAATCC
LT GGCGACAGATTATACCGTGC 581 GCGAGAGGAACACAAACCGG 581

CCGAATTCTGTTATATATGTC
EPEC eae —b
EAF CAGGGTAAAAGAAAGATGATAA 214 TATGGGGACCATGTATTATCA 313

TATGGGGACCATGTATTATCA
BFP AATGGTGCTTGCGCTTGCTGC 268 GCTACGGTGTTAATATCTCTGGCG 462

GCCGCTTTATCCAACCTGGTA

EHEC eae CAGGTCGTCGTGTCTGCTAAA 234 ACTGAAAGCAAGCGGTGGTG 691
TCAGCGTGGTTGGATCAACCT (O157:H7-specific)
SLTI TTTACGATAGACTTCTCGAC 223 GATGATCTCAGTGGGCGTTC 270
CACATATAAATTATTTCGCTC (SLT-I AND II)
SLTII As above TCTGAAACTGCTCCTGTGTA 270
Plasmid ACGATGTGGTTTATTCTGGA 223 CCGTATCTTATAATAAGACGGATGTTGG 223
CTTCACGTCACCATACATAT
EIEC ial CTGGATGGTATGGTGAGG 579 CCATCTATTAGAATACCTGTG 579

GGAGGCCAACAATTATTTCC
EAEC Plasmid CTGGCGAAAGACTGTATCAT 576 None

CAATGTATAGAAATCCGCTGTT
a
Each primer is written 59-39. See the text for abbreviations and discussion.
b
No oligonucleotide primers have yet been described which will detect specifically all human EPEC strains. (See reference 234.)
Pathogenesis (Fig. 4A) (622). The B subunits are arranged in a ring or

“doughnut” and bind strongly to the ganglioside GM1 and
ETEC strains are generally considered to represent a patho- weakly to GD1b and some intestinal glycoproteins (643). The
genic prototype: the organisms colonize the surface of the A subunit is responsible for the enzymatic activity of the toxin
small bowel mucosa and elaborate their enterotoxins, giving and is proteolytically cleaved to yield A1 and A2 peptides
rise to a net secretory state. Some investigators have reported joined by a disulfide bond. Two closely related variants of LT-I
that ETEC strains may exhibit limited invasiveness in cell cul- which exhibit partial antigenic cross-reactivity have been de-
tures, but this has not been demonstrated in vivo (189, 190). scribed. These variants are called LTp (LTp-I) and LTh
ETEC strains cause diarrhea through the action of the en- (LTh-I) after their initial discovery in strains isolated from pigs
terotoxins LT and ST. These strains may express an LT only, or humans, respectively. The genes encoding LT (elt or etx)
an ST only, or both an LT and an ST. These toxins have
reside on plasmids that also may contain genes encoding ST
recently been reviewed (291, 293, 295, 296, 480, 589), and the
and/or colonization factor antigens (CFAs).
reader is referred to these sources for primary references.
After binding to the host cell membranes, the toxin is endo-
Heat-labile toxins. The LTs of E. coli are oligomeric toxins
cytosed and translocated through the cell in a process involving
that are closely related in structure and function to the cholera
enterotoxin (CT) expressed by Vibrio cholerae (596). LT and trans-Golgi vesicular transport (378). The cellular target of LT
CT share many characteristics including holotoxin structure, is adenylate cyclase located on the basolateral membrane of
protein sequence (ca. 80% identity), primary receptor identity, polarized intestinal epithelial cells. The A1 peptide has an
enzymatic activity, and activity in animal and cell culture as- ADP-ribosyltransferase activity and acts by transferring an
says; some differences are seen in toxin processing and secre- ADP-ribosyl moiety from NAD to the alpha subunit of the
tion and in helper T-lymphocyte responses (153). There are GTP-binding protein, GS, which stimulates adenylate cyclase
two major serogroups of LT, LT-I and LT-II, which do not activity. ADP-ribosylation of the GSa subunit results in ade-
cross-react immunologically. LT-I is expressed by E. coli strains nylate cyclase being permanently activated, leading to in-
that are pathogenic for both humans and animals. LT-II is creased levels of intracellular cyclic AMP (cAMP). cAMP-
found primarily in animal E. coli isolates and rarely in human dependent protein kinase (A kinase) is thereby activated,
isolates, but in neither animals nor humans has it been asso- leading to supranormal phosphorylation of chloride channels
ciated with disease. Unless otherwise distinguished by Roman located in the apical epithelial cell membranes. The major
numerals, the term LT below refers to the LT-I form. chloride channel activated by LT and CT is CFTR (589), the
(i) LT-I. LT-I is an oligomeric toxin of ca. 86 kDa composed ion channel that is defective in cystic fibrosis. The net result is
of one 28-kDa A subunit and five identical 11.5-kDa B subunits stimulation of Cl2 secretion from secretory crypt cells and
inhibition of NaCl absorption by villus tip cells. The increased GM1 (229). As noted above, there is no evidence that LT-II is
luminal ion content draws water passively through the para- associated with human or animal disease.
cellular pathway, resulting in osmotic diarrhea. Heat-stable toxins. In contrast to the large, oligomeric LTs,
Although the stimulation of Cl2 as a result of increased the STs are small, monomeric toxins that contain multiple
intracellular levels of cAMP is the classical explanation for the cysteine residues, whose disulfide bonds account for the heat
mechanism by which LT and CT cause diarrhea, there is instability of these toxins. There are two unrelated classes of STs
creasing evidence, obtained mostly with CT, that the secretory that differ in structure and mechanism of action. Genes for
response to these toxins is considerably more complex (re- both classes are found predominantly on plasmids, and some
viewed in reference 589). One alternative mechanism by which ST-encoding genes have been found on transposons. STa (also
these toxins could act involves prostaglandins of the E series called ST-I) toxins are produced by ETEC and several other
(PGE1 and PGE2) and platelet-activating factor. Synthesis and gram-negative bacteria including Yersinia enterocolitica and V.
release of arachidonic acid metabolites such as prostaglandins cholerae non-O1. STa has about 50% protein identity to the
and leukotrienes can stimulate electrolyte transport and intes- EAST1 ST of EAEC, which is described further below. It has
tinal motility. A second alternative mechanism involves the recently been reported (564, 706) that some strains of ETEC
enteric nervous system (ENS), which regulates intestinal mo- may also express EAST1 in addition to STa. STb has been
tility and ion secretion. Serotonin and vasoactive intestinal found only in ETEC.
polypeptide, both of which can stimulate intestinal epithelial (i) STa. The mature STa is an 18- or 19-amino-acid peptide

cell secretion via the ENS, are released into the human small with a molecular mass of ca. 2 kDa. There are two variants,
bowel after treatment with CT (186). A third potential mech- designated STp (ST porcine or STIa) and STh (ST human or
anism could involve a mild intestinal inflammatory response STIb), after their initial discovery in strains isolated from pigs
due to CT and LT. CT has been reported to stimulate produc- or humans, respectively. Both variants can be found in human
tion of the proinflammatory cytokine interleukin-6 (IL-6), ETEC strains. These two variants are nearly identical in the 13
thereby activating the enteric immune system and potentially residues that are necessary and sufficient for enterotoxic activ-
generating arachidonic acid metabolites that stimulate secre- ity, and of these 13 residues, 6 are cysteines which form three
tion (433). These alternative secretory mechanisms are sup- intramolecular disulfide bonds. STa is initially produced as a
ported by a variety of in vitro and in vivo data, and one or more 72-amino-acid precursor (pre-pro form) that is cleaved by sig-
of them could act in concert with the classic mode of action nal peptidase 1 to a 53-amino-acid peptide (533). This form is
involving cAMP in causing diarrhea due to LT and CT. The transported to the periplasm, where the disulfide bonds are
similarity of LT and CT is considered sufficiently high to ex- formed by the chromosomally encoded DsbA protein (708).
trapolate mechanistic similarities between the two toxins, and An undefined protease processes the pro-STa to the final 18-
the validity of these assumptions has proven largely correct, or 19-residue mature toxin which is released by diffusion across
with the exception of the failure of LT to release serotonin the outer membrane.
(660). However, observations made to date for secondary ef- The major receptor for STa is a membrane-spanning enzyme
fects of CT have not all been demonstrated for LT, nor has the called guanylate cyclase C (GC-C), which belongs to a family
clinical relevance of these secondary secretory effects been of receptor cyclases that includes the atrial natriuretic peptide
substantiated. receptors GC-A and GC-B (152, 670). Additional receptors for
CT and LT have been shown as well to decrease the absorp- STa may exist (292, 410), but GC-C is the only receptor iden-
tion of fluid and electrolytes from the intestinal lumen (200). tified definitively. GC-C is located in the apical membrane of
Muller et al. have reported that both CT and LT induce intestinal epithelial cells, and binding of ligands to the extra-
cAMP-dependent inhibition of the H1/peptide cotransporter cellular domain stimulates the intracellular enzymatic activity.
in the human intestinal cell line Caco-2 (456). Interestingly, A mammalian hormone called guanylin is the endogenous
since the H1/peptide cotransporter does not possess sites for agonist for GC-C (106). Guanylin is a 15-amino-acid peptide
phosphorylation by protein kinase A (PKA), the authors pro- which contains four cysteines and is less potent than STa in
pose that the effect is mediated through PKC. This hypothesis activating GC-C. Guanylin is presumed to play a role in normal
would suggest another novel mechanism of CT and LT and gut homeostasis, and GC-C is apparently used opportunisti-
requires substantiation in other systems. cally by STa to cause diarrhea.
In addition to its enterotoxic properties, LT has the ability to Binding of STa to GC-C stimulates GC activity, leading to
serve as a mucosal adjuvant. Mutants of LT which retain ad- increased intracellular cGMP levels (138, 446, 589) (Fig. 4B).
juvanticity while eliminating the ADP-ribosyltransferase activ- This activity leads ultimately to stimulation of chloride secre-
ity have been constructed (153, 167, 460). Mice immunized tion and/or inhibition of sodium chloride absorption, resulting
orally or intranasally with ovalbumin or fragment C of tetanus in net intestinal fluid secretion. The intermediate steps in-
toxin together with the mutant LTs have developed higher volved in this process are controversial, and roles for both
levels of serum and local antibodies to these antigens than cGMP-dependent kinases and cAMP-dependent kinases have
when the antigens are delivered without LT. This property been reported (589). Ultimately, the CFTR chloride channel is
could simplify vaccine development and administration for a activated, leading to secretion of Cl2 ions into the intestinal
variety of pathogens by permitting oral or nasal, rather than lumen. In contrast to the 15- to 60-min lag time needed for LT
parenteral, administration of antigens. to translocate to and activate the basolateral adenylate cyclase
(ii) LT-II. The LT-II serogroup of the LT family shows 55 to complex, STa acts much faster due to the apical location of its
57% identity to LT-I and CT in the A subunit but essentially no cyclase receptor. Alternative mechanisms of action for STa
homology to LT-I or CT in the B subunits (229, 271, 518, 589, involving prostaglandins, calcium, and the ENS have been pro-
612). Two antigenic variants, LT-IIa and LT-IIb, which share posed (477, 478), but the evidence for the involvement of these
71 and 66% identity in the predicted A and B subunits, respec- factors is inconsistent. The secretory response to STa may also
tively, have been described. LT-II increases intracellular involve phosphatidylinositol and diacylglycerol release, activa-
cAMP levels by similar mechanisms to those involved with tion of PKC, elevation of intracellular calcium levels, and mi-
LT-I toxicity, but LT-II uses GD1 as its receptor rather than crofilament (F-actin) rearrangement (reviewed in reference 589).
FIG. 2. Various morphologies of diarrheagenic E. coli fimbriae as seen by transmission electron microscopy. (A) Rigid fimbrial morphology illustrated by ETEC
fimbriae CS1 (labelled CFA/II in the figure). The diameter of individual fimbriae is ca. 7 nm. (B) Flexible fibrillar morphology exemplified by the CS3 component of
CFA/II (arrow). Note the typical narrow diameter, ca. 2 to 3 nm, and the coiled appearance. (C) Electron micrograph showing the EPEC bundle-forming pilus expressed
by strain E2348/69. Bar, 0.35 mm. Reprinted from reference 245 with permission of the publisher.

FIG. 2— Continued.
(ii) STb. STb is associated primarily with ETEC strains iso- antigens have been characterized (Table 3), although the fim-
lated from pigs, although some human ETEC isolates express- briae of some ETEC strains have yet to be identified and are
ing STb have been reported. STb is initially synthesized as a only presumed to exist. Clearly, the antigenic heterogeneity
71-amino-acid precursor protein, which is processed to a ma- conferred by the existence of multiple fimbrial antigens is an
ture 48-amino-acid protein with a molecular weight of 5.1 kDa obstacle to effective vaccine development.
(23, 171). The STb protein sequence has no homology to that ETEC fimbriae confer the species specificity of the patho-
of STa, although it does contain four cysteine residues which gen. For example, ETEC strains expressing K99 are patho-
form disulfide bonds (23). Unlike STa, STb induces histologic genic for calves, lambs and pigs, whereas K88-expressing or-
damage in the intestinal epithelium, consisting of loss of villus ganisms are able to cause disease only in pigs (109). Human
epithelial cells and partial villus atrophy. The receptor for STb ETEC strains possess their own array of colonization fimbriae,
is unknown, although it has been suggested recently that the the CFAs (150). The terminology of the CFAs is confusing and
toxin may bind nonspecifically to the plasma membrane prior inconsistent. However, a uniform scheme has been proposed
to endocytosis (115). Unlike the chloride ion secretion elicited which would number each putative CFA consecutively accord-
by STa, STb stimulates the secretion of bicarbonate from in- ing to the year of its initial description (230); the number would
testinal cells (589). STb does not stimulate increases in intra- be preceded by the initials CS, for coli surface antigen. We
cellular cAMP or cGMP concentrations, although it does stim- support this proposed scheme, and it has been included in
ulate increases in intracellular calcium levels from extracellular Table 3.
sources (170). STb also stimulates the release of PGE2 and The CFAs can be subdivided based on their morphologic
serotonin, suggesting that the ENS may also be involved in the characteristics. Three major morphologic varieties exist: rigid
secretory response to this toxin (228, 294). rods, bundle-forming flexible rods, and thin flexible wiry struc-
Colonization factors. The mechanisms by which ETEC tures. CFA/I, the prototype rigid rod-shaped fimbria, is com-
strains adhere to and colonize the intestinal mucosa have been posed of a single protein assembled in a tight helical configu-
the subject of intensive investigation (for recent reviews, see ration (308). CFA/III is a bundle-forming pilus with homology
references 109, 149, 230, and 697). To cause diarrhea, ETEC to the type 4 fimbrial family (633, 634). CFA/II and CFA/IV
strains must first adhere to small bowel enterocytes, an event are in fact composed of multiple distinct fimbrial structures:
mediated by surface fimbriae (also called pili). Transmission CFA/II producers express the flexible CS3 structure either
electron microscopy of ETEC strains typically reveals many alone or in association with the rod-shaped CS1 or CS2 (389,
fimbriae peritrichously arranged around the bacterium; often, 597); CFA/IV producers express CS6 in conjunction with CS4
multiple fimbrial morphologies can be visualized on the same or CS5 (109, 363). A large number of other, less common
bacterium (389) (Fig. 2B). A large number of ETEC fimbrial adhesins have also been found in ETEC strains (150), yet

FIG. 3. Pathogenic schemes of diarrheagenic E. coli. The six recognized categories of diarrheagenic E. coli each have unique features in their interaction with
eukaryotic cells. Here, the interaction of each category with a typical target cell is schematically represented. It should be noted that these descriptions are largely the
result of in vitro studies and may not completely reflect the phenomena occurring in infected humans. See the text for details.
epidemiologic studies suggest that CFA/I, CFA/II, or CFA/IV Epidemiology

is expressed by approximately 75% of human ETEC strains ETEC strains are associated with two major clinical syn-
worldwide (697). A newly described ETEC fimbria, designated dromes: weanling diarrhea among children in the developing
Longus, has been found on a large proportion of human ETEC world, and traveler’s diarrhea. The epidemiologic pattern of
(244, 246). ETEC disease is determined in large part by a number of
The genetics of CFAs have been studied extensively, and factors: (i) mucosal immunity to ETEC infection develops in
these studies have served to illuminate models for fimbrial exposed individuals; (ii) even immune asymptomatic individu-
expression, protein secretion and translocation, and the assem- als may shed large numbers of virulent ETEC organisms in the
bly of bacterial organelles (Fig. 5). CFA genes are usually stool; and (iii) the infection requires a relatively high infectious
encoded on plasmids, which typically also encode the entero- dose (175). These three features create a situation in which
toxins ST and/or LT (150). Typical fimbrial gene clusters con- ETEC contamination of the environment in areas of endemic
sist of a series of genes encoding a primary fimbrial subunit infection is extremely prevalent, and most infants in such areas
protein and accessory proteins which are required for process- will encounter ETEC upon weaning. The percentage of cases
ing, secretion, and assembly of the fimbrial structure itself of sporadic endemic infant diarrhea which are due to ETEC
(150, 308, 319, 370). The pilin structural subunit is usually the usually varies from 10 to 30% (12, 209, 298, 385, 406, 581, 654).
predominant immunogen and is thus subject to the greatest School-age children and adults typically have a very low inci-
antigenic pressure. Pilin subunits accordingly exhibit the great- dence of symptomatic ETEC infection. Characteristically, ST-
est sequence variation; however, the N termini of the subunit producing ETEC strains cause the majority of endemic cases
proteins, as well as the accessory proteins, are generally at least (12, 385).
partially conserved. This phenomenon is believed to reflect Epidemiologic investigations have implicated contaminated
structure-function requirements (370). Although the actual food and water as the most common vehicles for ETEC infec-
protein adhesin of some E. coli fimbriae (such as pap and type tion (71, 73, 395, 700). Sampling of both food and water
1 fimbriae) is a tip protein distinct from the structural protein sources from areas of endemic infection have demonstrated
comprising the stalk, the adhesin of diarrheagenic E. coli fim- strikingly high rates of ETEC contamination (550, 700); this is
briae is generally the stalk protein itself. not unexpected given that 108 CFU of ETEC with buffer must

FIG. 4. Classic mechanisms of action of ETEC toxins (see the text for details and additional proposed mechanisms). (A) LT-I. The LT holotoxin, consisting of one
A subunit and five B subunits, is internalized by epithelial cells of the small bowel mucosa via endocytosis. The A1, or catalytic, subunit translocates through the vacuolar
membrane and passes through the Golgi apparatus by retrograde transport. In the figure, the A subunit is shown passing through the B subunit ring, but this may not
be the case in vivo. A1 catalyzes the ADP-ribosylation of arginine 201 of the a subunit of Gs-protein (which may be apically located); the ADP-ribosylated G-protein
activates adenylate cyclase, which elicits supranormal levels of intracellular cAMP. cAMP is an intracellular messenger which regulates several intestinal epithelial cell
membrane transporters and other host cell enzymes, as well as having effects on the cytoskeleton. The activation of the cAMP-dependent A kinase results in
phosphorylation of apical membrane transporters (especially the cystic fibrosis transmembrane conductance regulator), resulting in secretion of anions (predominantly
Cl2 by a direct effect, and HCO32 indirectly) by crypt cells and a decrease in absorption of Na1 and Cl2 by absorptive cells. cAMP may also have important effects
on basolateral transporters and on intracellular calcium levels, both of which may increase the magnitude of the effects on fluid and ion transport. (B) STa. Less is known
about the action of ST than of LT. ST is thought to act by binding the ST membrane receptor, GC-C. Activation of GC-C results in increased levels of intracellular
cGMP. cGMP exerts its effects in increasing chloride secretion and decreasing NaCl absorption by activating the cGMP-dependent kinase (G-kinase) and/or the cAMP
dependent kinase (A-kinase). Other effects of STa in inducing fluid secretion have also been postulated (see the text).
be given to induce high attack rates in volunteers (175, 383). ETEC traveler’s diarrhea occurs most commonly in warm and
Thus, fecal contamination of water and food sources is the wet months and among first-time travelers to the developing
principal reason for the high incidence of ETEC infection world (21). Traveler’s diarrhea is usually contracted from con-
throughout the developing world, and the institution of appro- taminated food and water (70, 422, 700).
priate sanitation is the cornerstone of preventive efforts against
this infection. Clinical Considerations
ETEC infections in areas of endemic infection tend to be
clustered in warm, wet months, when multiplication of ETEC The clinical characteristics of ETEC disease are consistent
in food and water is most efficient (381). Person-to-person with the pathogenetic mechanisms described above. Similar
transmission was not found to occur during a study of ETEC- features of the illness have been demonstrated in both volun-
infected volunteers housed side by side with volunteers en- teers and patients in areas of endemic infection. The illness is
rolled in an evaluation of influenza vaccine candidates (388). typically abrupt in onset with a short incubation period (14 to
Although ETEC infection occurs most frequently in infants, 50 h) (175, 459). The diarrhea is watery, usually without blood,
immunologically naive adults are susceptible (this stands in mucus, or pus; fever and vomiting are present in a minority of
contrast to EPEC infection, as described below). Indeed, patients (175, 381). ETEC diarrhea may be mild, brief, and
ETEC is the predominant etiologic agent causing traveler’s self-limiting or may result in severe purging similar to that seen
diarrhea among adults from the developed world visiting areas in V. cholerae infection (383).
where ETEC infection is endemic (21, 70, 174, 422). Studies Most life-threatening cases of ETEC diarrhea occur in
suggest that 20 to 60% of such travelers experience diarrhea; weanling infants in the developing world. Even though the
typically, 20 to 40% of cases are due to ETEC. Predictably, administration of antibiotics to which ETEC strains are sus-
TABLE 3. CFAs of human ETEC strains purified fimbriae, attenuated ETEC strains, and attenuated
Salmonella, Shigella, and V. cholerae strains expressing ETEC
Original
designation
CS designation Diameter (nm) Reference(s) antigens (reviewed in references 626 and 630). An oral cholera
vaccine containing killed V. cholerae and purified CT B subunit
Rigid rods has been reported to provide protection against traveler’s di-
CFA/I CFA/I 7 321 arrhea due to ETEC (511). This protection is presumably due
CS1 CS1 7 225, 320, 513 to the antigenic similarity between LT and CT, although this
CS2 CS2 7 226
CS4 CS4 6 698
would not explain the protection against ETEC strains express-
PCFO159 CS12 7 576 ing ST. Development of an ETEC vaccine with broad protec-
PCFO166 CS14 7 427 tion is greatly complicated by the numerous intestinal coloni-
CS17 CS17 7 428 zation factors expressed by ETEC.
PCFO20 CS18 7 680
CS19 CS19 7 230 Detection and Diagnosis
CS20 CS20 7 671
Detection of ETEC has long relied on detection of the
Bundle-forming
enterotoxins LT and/or ST. ST was initially detected in a rabbit
CFA/III CS8 7 634
ligated ileal loop assay (193), but the expense and lack of

Longus CS21 7 244
standardization caused this test to be replaced by the suckling-
Fibrillar mouse assay (236), which became the standard test for the
CS3 CS3 2–3 86 presence of STa for many years. The suckling-mouse assay
CS5 CS5 5 127, 411 entails the measurement of intestinal fluid in CD4 infant mice
PCFO148 CS11 3 362 after percutaneous injection of culture supernatants.
PCFO9 CS13 285 Several immunoassays have been developed for detection of
ST, including a radioimmunoassay (237) and an enzyme-linked
Nonfimbrial
immunosorbent assay (ELISA) (144) (available from Denka
CS6 CS6 698
2230 CS10 147 Seiken, Co. Ltd., Tokyo, Japan). Both of these tests correlate
8786 CS15 25 well with results of the suckling-mouse assay and require sub-
stantially less expertise (144).
The traditional bioassay for detection of LT involves the use
of cell culture, either the Y1 adrenal cell assay or the Chinese
ceptible has been shown to decrease both the duration of hamster ovary (CHO) cell assay. In the Y1 assay, ETEC cul-
diarrhea and the intensity of ETEC excretion (72, 173), effec-
tive agents may not be available in areas where the incidence is
high; moreover, antibiotic resistance in ETEC strains is an
emerging problem, and in many areas (174) effective agents
which are safe for children are not readily available. It should
be kept in mind, therefore, that the cornerstone of manage-
ment of ETEC infection is to maintain a normal hydration
status. Oral rehydration therapy is often lifesaving in infants
and children with ETEC diarrhea.
Travelers to the developing world should also be counseled
on the need to maintain hydration when they experience diar-
rhea. In addition, bismuth subsalicylate or loperamide is effec-
tive in decreasing the severity of diarrhea (21); the latter
should not be administered to patients with fever or dysentery
unless antibiotics are also given. Antibiotics given empirically
for traveler’s diarrhea can shorten the duration of the episode
(191). Currently, fluoroquinolones (e.g., ciprofloxacin, nor-
floxacin, and ofloxacin) are the most commonly recommended
agents, since increasing antimicrobial resistance to traditional
agents has been documented in several areas (173, 174).
FIG. 5. Genetics of E. coli fimbriae. Genes required for the expression of
Travelers to developing areas are often concerned with the functional pili are characteristically linked in gene clusters. The genetic organi-
development of traveler’s diarrhea and may seek a means of zation of these clusters is illustrated for ETEC fimbriae CS1, CFA/I, CS3, and
preventing it. Doxycycline and trimethoprim-sulfamethoxazole CS6, and for members of the Dr family, found in DAEC and EAEC. Italicized
have been shown to be effective in this regard, although in- terms in parentheses represent the gene designations, to be followed by the
specific letter under the corresponding arrow to the right. Arrows of similar fill
creasing resistance would suggest that fluoroquinolones ad- pattern have genetic and functional homology; black arrows represent structural
ministered once daily would be more effective (280). However, subunits. The known functions of the genes in the Dr cluster are listed below the
the growing problem of antibiotic resistance and the possibility corresponding genes. These functions can be extrapolated to arrows of similar fill
of adverse effects from antimicrobial agents weigh strongly pattern in the CS3 and CS6 gene cluster. The usher and chaperone genes from
the Dr, CS6, and CS3 clusters have homology to the genes serving these functions
against recommending antimicrobial prophylaxis routinely. in pap fimbriae: usher proteins are OMPs which serve as pores for the transport
Rather, experts have recommended (i) avoiding potentially and assembly of the fimbrial shaft; fimbrial chaperones bind to the fimbrial
contaminated food and drink while traveling, (ii) bismuth sub- subunit proteins in the periplasmic space and prevent premature folding and
salicylate given four times daily, and (iii) the use of antibiotics degradation. CS1 and CFA/I accessory genes, required for assembly and trans-
port of the fimbriae, are homologous to each other but not to CS3, CS6, or the
empirically if significant diarrhea develops (174). Dr family. CS6 has an unusual organization in that the first two genes of the
Oral vaccines against ETEC are being developed by a variety cluster apparently encode heterologous major subunit proteins (699); the signif-
of approaches including the use of killed whole cells, toxoids, icance of this feature is not yet understood.
ture supernatants are added to Y1 cells and the cells are Pathogenesis
examined for rounding (165). In the CHO cell assay, LT will
cause elongation of the CHO cells (265). Immunologic assays Attaching-and-effacing histopathology. The hallmark of in-
are easier to implement in clinical laboratories and include the fections due to EPEC is the attaching-and-effacing (A/E) his-
traditional Biken test (297) as well as newer immunologic topathology, which can be observed in intestinal biopsy speci-
methods such as ELISA (709), latex agglutination (304), and mens from patients or infected animals and can be reproduced
two commercially available tests, the reversed passive latex in cell culture (18, 314, 358, 453, 524, 547, 616, 640, 667, 669)
agglutination test (582) and the staphylococcal coagglutination (Fig. 6). This striking phenotype is characterized by effacement
test (116). Both of the commercially available tests are reliable of microvilli and intimate adherence between the bacte-
and easy to perform (613). rium and the epithelial cell membrane. Marked cytoskeletal
ETEC strains were among the first pathogenic microorgan- changes, including accumulation of polymerized actin, are seen
isms for which molecular diagnostic techniques were devel- directly beneath the adherent bacteria; the bacteria sometimes
oped. As early as 1982 (455), DNA probes were found to be sit upon a pedestal-like structure. These pedestal structures
useful in the detection of LT- and ST-encoding genes in stool can extend up to 10 mm out from the epithelial cell in pseu-
and environmental samples. Since that time, several advances dopod-like structures (453). This lesion is quite different from
in ETEC detection have been made, but genetic techniques the histopathology seen with ETEC strains and V. cholerae, in
continue to attract the most attention and use. It should be which the organisms adhere in a nonintimate fashion without

stressed that there is no perfect test for ETEC: detection of causing microvillous effacement or actin polymerization. Al-
colonization factors is impractical because of their great num- though earlier studies had also reported this histopathology, it
ber and heterogeneity; detection of LT and ST defines an was not until the report by Moon et al. (453) that the pheno-
ETEC isolate, yet many such isolates will express colonization type became widely associated with EPEC and the term “at-
factors specific for animals and thus lack human pathogenicity. taching and effacing” was coined.
The LT polynucleotide probe provides good sensitivity and The initial observation by Knutton et al. (359) that the com-
specificity when labeled with radioisotopes (373, 455) or with position of the A/E lesion contained high concentrations of
enzymatic, nonisotopic detection systems (528). Several differ- polymerized filamentous actin (F-actin) led to the develop-
ent protocols have been published in which nonisotopic label- ment of the fluorescent-actin staining (FAS) test. In this test,
ing methods have proven useful for LT detection (2, 117, 718); fluorescein isothiocyanate (FITC)-labeled phalloidin binds
specifically to filamentous actin in cultured epithelial cells di-
we now use a highly reliable alkaline phosphatase-based de-
rectly beneath the adherent bacteria. Prior to the development
tection system (Blue Gene; Gibco-BRL) for use in polynucle-
of this test, the A/E histopathology could be detected only by
otide probe colony blot hybridization.
the use of electron microscopy and intact animals or freshly
ST polynucleotide probes have had problems of poor sensi-
isolated intestinal epithelial cells. Besides providing a diagnos-
tivity and specificity, presumably because of the small size of
tic test for EPEC strains and other organisms capable of caus-
the gene. For this reason, oligonucleotide probes which are
ing this histopathology, the FAS test enabled the screening of
generally more sensitive and specific for ST detection have clones and mutants, leading to the identification of the bacte-
been developed (581) (Table 2 lists the nucleotide sequences rial genes involved in producing this pathognomonic lesion.
of oligonucleotides used for probing and PCR of diarrheagenic In addition to F-actin, the composition of the A/E lesion
E. coli strains). An LT oligonucleotide has also been developed includes other cytoskeletal components such as a-actinin, talin,
(581), but this reagent has relatively few advantages over an ezrin, and myosin light chain (205). At the tip of the pedestals
enzymatically detected LT fragment probe. Recently, a triva- beneath the plasma membrane are located proteins that are
lent oligonucleotide probe has been proposed which may be of phosphorylated on a tyrosine residue in response to EPEC
use in detecting the genes encoding LT, ST, and the EHEC infection (see below). The formation of the pedestal is a dy-
Shiga toxin genes (see below); this probe shows promise in an namic process, and video microscopy shows that these EPEC
early report (44). ETEC strains are particularly amenable to pedestals can bend and undulate, alternatively growing longer
stool blot hybridization because of the large number of organ- and shorter while remaining tethered in place on the cell sur-
isms typically shed in the stools of infected individuals (615). face (557). Some of the attached EPEC organisms can actually
Several PCR assays for ETEC are quite sensitive and spe- move along the surface of the cultured epithelial cell, reaching
cific (177, 374, 492, 581, 615, 654) when used directly on clinical speeds up to 0.07 mm/s in a process driven by polymerization of
samples or on isolated bacterial colonies. A useful adaptation actin at the base of the pedestal. This motility resembles that
of PCR is the “multiplex” PCR assay (374, 615), in which seen with Listeria spp. (650) inside eukaryotic cells, except that
several PCR primers are combined with the aim of detecting the motile EPEC organisms are located extracellularly. The
one of several different diarrheagenic E. coli pathotypes in a significance of this motility observed in vitro to the pathogen-
single reaction. After multiplex PCR, various reaction prod- esis of disease caused by EPEC is unknown. Similar A/E le-
ucts can usually be differentiated by product size, but a sec- sions are seen in animal and cell culture models of EHEC (see
ond detection step (e.g., nonisotopic probe hybridization) below) and Hafnia alvei isolated from children with diarrhea
is generally performed to identify the respective PCR (9, 11). However, only a small, highly conserved subset of H.
products definitively. alvei strains produce the A/E lesion (537, 538), and detailed
taxonomic studies suggest that the A/E-positive H. alvei strains
should not be included in the same species as the A/E-negative
ENTEROPATHOGENIC E. COLI H. alvei strains (537). The A/E lesion is also produced by
strains of Citrobacter rodentium (formerly Citrobacter freundii
EPEC is an important category of diarrheagenic E. coli biotype 4280) that cause murine colonic hyperplasia (although
which has been linked to infant diarrhea in the developing diarrhea is not seen in infection due to this species) (569). In
world. Once defined solely on the basis of O and H serotypes, addition to EPEC and EHEC, a variety of E. coli strains ca-
EPEC is now defined on the basis of pathogenetic character- pable of A/E have been isolated from rabbits (102), calves
istics, as described below. (206), pigs (717), and dogs (172). Thus, EPEC strains are the

FIG. 6. Characteristic EPEC A/E lesion observed in the ileum after oral inoculation of gnotobiotic piglets. Note the intimate attachment of the bacteria to the
enterocyte membrane with disruption of the apical cytoskeleton. The appearance of a bacterium sitting on a “pedestal” of cell membrane is quite characteristic.
Reprinted from reference 26 with permission of the publisher.
prototype of an entire family of enteric pathogens that produce Antiserum prepared against purified BFP significantly, al-
A/E lesions on epithelial cells. though not completely, reduced the localized adherence of
Three-stage model of EPEC pathogenesis. Multiple steps EPEC strain B171 (O111:NM) to HEp-2 cells. BFP are defi-
are involved in producing the characteristic A/E histopathol- nitely involved in bacterium-to-bacterium adherence in the
ogy. In 1992, Donnenberg and Kaper (158) proposed a three- localized adherence pattern, but there is no definitive proof
stage model of EPEC pathogenesis consisting of (i) localized that BFP mediates actual adherence to epithelial cells. The
adherence, (ii) signal transduction, and (iii) intimate adher- N-terminal sequence of the purified fimbriae revealed similar-
ence (Fig. 7). The temporal sequence of these stages is not ity to the TCP pilus of V. cholerae (242) and other members of
certain, and, indeed, the different stages may occur concur- the type IV fimbrial family. Donnenberg et al. (157) identified
rently. Nevertheless, this model has proven to be a robust one the structural gene encoding BFP (bfpA) by using a TnphoA
that can readily accommodate advances in our understanding mutant of E2348/69 which no longer conferred localized ad-
of EPEC pathogenesis that have been made since it was first herence. Subsequent genetic studies have revealed that a clus-
proposed. Additional details on this model can be found in ter of 13 genes on the EAF plasmid is required for the expres-
recent reviews (154, 159, 327). sion and assembly of BFP (609, 621). Many of these genes
(i) Localized adherence. As noted above, adherence to encode proteins with similarity to proteins required for type IV
HEp-2 cells was first described by Cravioto et al. for EPEC pilus biogenesis in other gram-negative pathogens such as V.
(139). Baldini et al. (26) showed that the ability of EPEC strain cholerae and Pseudomonas aeruginosa, but some BFP proteins
E2348/69 (O127:H6) to adhere in a localized pattern was de- have no obvious homologs. In addition, expression and assem-
pendent on the presence of a 60-MDa plasmid. Loss of this bly of BFP require the global regulator element of EPEC
plasmid led to loss of the LA phenotype, and transfer of this pathogenesis, Per (also called BfpTWV [see below]), and the
plasmid to nonadherent E. coli HB101 enabled this strain to chromosomal dsbA gene, encoding a periplasmic enzyme that
adhere to HEp-2 cells. This plasmid was therefore designated mediates disulfide bond formation (715).
the EPEC adherence factor (EAF) plasmid (see below), and a (ii) Signal transduction. Adherence of EPEC to epithelial
1-kb fragment from this region was developed as a diagnostic cells induces a variety of signal transduction pathways in the
DNA probe (the EAF probe) (27, 461). Although this probe eukaryotic cell. The bacterial genes responsible for this signal
proved to be extremely valuable in diagnosing EPEC (see transduction activity are encoded on a 35-kb pathogenicity
below) and elucidating the epidemiology of EPEC infections, island called the locus of enterocyte effacement (LEE), which
the exact nature of the adhesin mediating this adherence re- encodes a type III secretion system, multiple secreted proteins,
mained unknown for many years. and a bacterial adhesin called intimin (see below). Mutation of
The identity of the factor mediating localized adherence was the genes encoding the secreted proteins (espA, espB, and
reported in 1991 by Girón et al. (242), who described 7-nm- espD) or the genes encoding the type III secretion system (sep
diameter fimbriae produced by EPEC strains which tended to and esc) abolishes these multiple signalling events. However,
aggregate and form bundles, thereby suggesting the name none of these signalling events has been reproduced by the
“bundle-forming pilus” (BFP). These fimbriae were produced addition of EPEC culture supernatants to epithelial cells,
only under certain culture conditions, thereby accounting for thereby indicating that actual binding of the bacterium is nec-
the failure of previous investigators to identify them (584). essary for these changes.

FIG. 7. Three-stage model of EPEC pathogenesis. (A) The first stage is characterized by initial, relatively distant interaction of bacteria with the enterocyte layer.
This initial attachment is thought to be mediated by the bundle-forming pilus. (B) In the second stage, eae and other genes are activated, causing dissolution of the
normal microvillar structure. (C) In the third stage, the bacterium binds closely to the epithelial membrane via the protein intimin. Other bacterial gene products
mediate further disruption of the cytoskeleton and phosphorylation of cellular proteins. Modified from reference 158 with permission of the publisher.
Infection with EPEC induces increases in the intracellular phorylation on tyrosine residues (351, 544). The major ty-
calcium levels [Ca21i] in cultured epithelial cells to which they rosine-phosphorylated protein is a 90-kDa protein, called
are attached (30, 31, 179, 514). The calcium originates from Hp90, inserted into the epithelial cell membrane protein (544).
intracellular stores rather than from an influx of extracellular The tyrosine-phosphorylated proteins are part of the A/E le-
calcium, and buffering of intracellular calcium greatly reduces sion, and the distribution of the phosphorylated proteins is
the polymerization of actin and formation of the A/E lesion restricted to an area immediately beneath the adherent bacte-
(30, 179). The increase in [Ca21i] has been hypothesized to ria at the tip of the pedestals (545). Rosenshine et al. (545)
produce the cytoskeletal changes induced by EPEC via activa- have also shown that the tyrosine-phosphorylated Hp90 serves
tion of a calcium-dependent, actin-severing protein which as a receptor for the intimin adhesin (see below). Thus, the
could break down actin in the microvillus core (31). Further- signal transduction induced in epithelial cells by EPEC acti-
more, since increases in intracellular calcium can inhibit Na1 vates receptor binding activity as well as subsequent cytoskel-
and Cl2 absorption and stimulate chloride secretion in entero- etal rearrangements. The Hp90 protein has recently been
cytes (201, 202), these data also suggest that changes in [Ca21i] shown to be a bacterial protein called Tir (translocated intimin
may mediate the intestinal secretory response to EPEC. There receptor) (352a).
is evidence that calcium is released from 1,4,5-inositol trisphos- Experiments with polarized epithelial cells such as Caco-2 or
phate (IP3)-sensitive stores (31), and several investigators have T84 show that binding of EPEC results in a decrease in the
shown that binding of EPEC to cultured epithelial cells triggers transepithelial resistance of the monolayers (101, 514, 614).
the release of inositol phosphates including IP3 and IP4 in Although an initial report suggested that this drop in resistance
infected cells (179, 212, 360). The increase in the amount of involved a transcellular pathway (101), subsequent reports
inositol phosphates is consistent with the recently reported have demonstrated that the paracellular pathway with alter-
activation of phospholipase Cg1 by EPEC attached to epithe- ations in tight junctions is involved (514, 614). Buffering of
lial cells (351). increases in the intracellular calcium concentration completely
Adherence of EPEC to epithelial cells results in the phos- abrogated the change in resistance (614).
phorylation of several epithelial cell proteins on serine and In addition to the effects seen with intestinal epithelial cells,
threonine residues, the most prominent of which is myosin the signal transduction response to EPEC also includes migra-
light chain (407, 409). Activation of at least two kinases, PKC tion of polymorphonuclear leukocytes (PMNs). Using an in
and myosin light chain kinase, has been shown (28, 137, 408, vivo system in which polarized T84 intestinal epithelial cells are
712). Activation of PKC induces rapid changes in intestinal cocultured with PMNs, Savkovic et al. (565) showed that at-
water and electrolyte secretion in vivo and in vitro (532) and tachment of EPEC to the epithelial cells caused PMNs to cross
phosphorylation of myosin light chain can lead to increased the epithelial monolayer. Stimulation of PMN transmigration
permeability of tight junctions (408), thereby suggesting addi- across intestinal epithelial cells has been shown for invasive
tional potential mechanisms of diarrhea due to EPEC. organisms such as Salmonella spp. (429) but is unusual for a
Binding of EPEC to HeLa cells also induces protein phos- primarily noninvasive organism such as EPEC. Experimental
evidence supports a model in which the binding of EPEC to Expression of intimin in E. coli K-12 is not sufficient to
epithelial cells activates the eukaryotic transcription factor NF- mediate adherence to epithelial cells (314). However, E. coli
kB, which in turn upregulates the expression of the cytokine K-12 expressing intimin from EPEC strains or E. coli O157:H7
IL-8, which is a PMN chemoattractant (565, 566). Neutralizing can adhere to epithelial cells when the cells are preinfected
antibodies to IL-8 ablated ca. 50% of the chemotactic activity, with an eae mutant of EPEC (437). The eae mutant itself
suggesting that other epithelium-derived chemotactic factors cannot adhere intimately, but it can provide signals that trigger
are also stimulated by EPEC adherence. the epithelial cell to form a functional receptor to which K-12
(iii) Intimate adherence. Intimate adherence of EPEC to expressing intimin can adhere. Rosenshine et al. (545) have
epithelial cells is mediated by a 94- to 97-kDa outer membrane presented evidence that the EPEC receptor is a tyrosine-phos-
protein called intimin. The gene encoding intimin (eae, for E. phorylated 90-kDa membrane protein exposed on the surface
coli attaching and effacing) was first reported by Jerse et al. of epithelial cells. As discussed above, one of the signal trans-
(314), who screened TnphoA mutants of EPEC for loss of the duction events characteristic of EPEC adhering to epithelial
A/E phenotype by using the FAS test (the genes involved in cells is tyrosine phosphorylation of a 90-kDa protein (Tir).
EPEC pathogenesis are illustrated in Fig. 8). Although eae When this 90-kDa protein is not tyrosine-phosphorylated, it
mutants cannot adhere intimately to epithelial cells, they can cannot serve as a receptor. These investigators also showed
still induce the signal transduction activities described above that purified intimin protein fused to maltose binding protein
(212, 544, 565, 618). The eae gene is present in all EPEC, can bind to membranes extracted from cells preincubated with

EHEC, C. rodentium, and H. alvei strains capable of producing the eae mutant but not to membranes extracted from cells that
the A/E histopathology but is absent from E. coli strains in the have not been infected with this strain. In contrast to these
normal flora, ETEC strains, and other bacteria that do not results, Frankel et al. (217, 218) reported that purified intimin-
produce the A/E lesion. maltose binding protein fusions can adhere to epithelial cells
The predicted intimin protein has 31% identity and 50% that have not been preincubated with EPEC. These investiga-
similarity to the invasin protein of Yersinia species (301). Com- tors further report that intimin binds to b1 integrins (220),
parison of the intimin proteins of EPEC strain E2348/69 and which also serve as receptors for the invasin protein from
EHEC O157:H7 strain EDL933 reveals a striking pattern of Yersinia species (379). The reason for these discrepant results
sequence conservation among intimin proteins (711). Al- is not clear, but it is possible that intimin can bind to more than
though the overall protein identity is 83%, the sequence diver- one receptor, and the question of which receptor is relevant for
gence is concentrated in the C-terminal region. The first 75% adherence to intestinal tissue remains to be answered.
of the protein (i.e., the first 704 amino acid residues starting Secreted proteins. A secreted enterotoxin that would explain
from the N terminus) has 94% identity, while the remaining the mechanism of diarrhea due to EPEC has been unsuccess-
25% of the residues has only 49% identity (711). The highly fully sought for many years (542). It was recently discovered by
divergent C-terminal region is the portion of the molecule that three independent groups that EPEC strains can secrete pro-
binds to receptors on the epithelial cell (217), and the different teins into the culture supernatant if grown in cell culture media
intimin sequences can confer different colonization patterns (273, 309, 350). These proteins, called Esps (for EPEC-se-
within the intestine (see the section on EHEC, below). There creted proteins), are also produced during the course of dis-
is a growing family of intimin proteins, and sequences have ease, since volunteers experimentally infected with EPEC pro-
been determined for at least nine intimin proteins from EPEC duce antibodies against a number of these proteins (309).
(8, 314, 398, 687), EHEC (8, 43, 398, 711), C. rodentium (570), However, in contrast to conventional enterotoxins, addition of
H. alvei (217), and E. coli strains pathogenic for rabbits and purified preparations of these secreted proteins has no effect
swine (8). The intimin proteins from these different pathogens on epithelial cells; only when the proteins are presented to the
are referred to as IntEPEC, IntO26 (from an O26 E. coli strain), target epithelial cell by an attached EPEC can they bring about
IntHA (from Hafnia alvei), etc. The overall pattern for these the various signal transduction changes in the epithelial cell
sequences shows high conservation in the N-terminal region outlined above.
and variability in the C-terminal region. At least four proteins are secreted extracellularly by EPEC,
The role of intimin in human disease was demonstrated by and three of these are essential for the A/E histopathology.
studies in volunteers, who ingested an isogenic eae null mutant The proteins that are essential for the A/E phenotype and their
of E2348/69 (161). Diarrhea was seen in 11 of 11 volunteers apparent molecular masses on sodium dodecyl sulfate-polyac-
ingesting the wild-type E2348/69 compared to 4 of 11 volun- rylamide gel electrophoresis are EspA (25 kDa) (352), EspB
teers ingesting the isogenic mutant (P 5 0.002). These results (38 kDa; formerly called EaeB) (164, 211, 273, 350), and EspD
indicate that the eae gene is essential for full virulence of (40 kDa) (371). Mutation of the espA, espB, or espD gene
EPEC strain E2348/69 but that additional virulence factors are abolishes the signal transduction in epithelial cells produced by
clearly required for disease. Prior to the discovery of the eae wild-type EPEC and the A/E histopathology. A fourth protein
gene, Levine et al. (386) reported that a 94-kDa outer mem- of ca. 110 kDa, called EspC, is homologous to members of the
brane protein (OMP) engendered a strong antibody response autotransporter protein family, which includes IgA proteases
in volunteers experimentally infected with EPEC. Subsequent of Neisseria gonorrhoeae and Haemophilus influenzae, Tsh pro-
studies showed that this immunogenic 94-kDa OMP is intimin, tein produced by avian pathogenic E. coli, SepA of Shigella
the product of the eae gene (312). Interestingly, in the volun- flexneri, and AIDA-I of DAEC (617). Mutation of the espC
teer studies conducted by Levine et al. (386) with 10 volun- gene does not affect signal transduction, A/E histopathology,
teers, the 9 who became ill upon challenge had no preexisting or any other obvious pathogenic phenotype of EPEC.
antibodies to the 94-kDa OMP. In the other volunteer, who did The EspA, EspB, and EspD proteins are translated without
not become ill, antibodies to intimin were present in sera a conventional N-terminal signal peptide (leader sequence).
collected prior to challenge. This result hints that intimin may Jarvis et al. (309) showed that EPEC possesses a type III
play a role in protective immunity to disease due to EPEC. protein secretion system similar to other specialized protein
Secretory immunoglobulin A (IgA) to a 94-kDa OMP of secretion systems found in a variety of gram-negative human,
E2348/69 was also found in breast milk from women in a rural animal, and plant pathogens. The type III secretion systems are
Mexican village (143). responsible for secretion and translocation of critical virulence

FIG. 8. Genes involved in EPEC pathogenesis. Genes involved in the pathogenesis of EPEC-induced diarrhea are presented in schematic fashion. Chromosomal
virulence genes are clustered within the LEE, which encodes a type III secretory apparatus as well as intimin and a cluster of secreted effector proteins. The EAF
plasmid encodes the BFP as well as a cluster of genes required for normal expression of BFP and intimin.
determinants such as Shigella Ipa proteins, Yersinia Yops, and called LEE (for locus of enterocyte effacement), is not present
proteins involved in invasion by Salmonella spp. (231, 439, in E. coli strains in the normal flora, E. coli K-12, or ETEC.
674). In EPEC, the genes encoding this secretion system were Sequences homologous to the EPEC sequence are also found
initially named sep (for secretion of EPEC proteins), and at in other Enterobacteriaceae that cause the A/E phenotype, in-
least nine sep genes encoding this protein secretion system cluding EHEC, the rabbit diarrheal pathogen RDEC-1, diar-
have been discovered (309, 430). The nomenclature for these rheagenic H. alvei, and C. rodentium. The LEE region of EPEC
genes has recently been revised to correspond with type III E2348/69 is inserted into the E. coli K-12 chromosome at ca. 82
secretion systems in Yersinia and other species (188). Those min, where the tRNA for selenocysteine (selC) is located.
EPEC genes with homologs to Yersinia ysc genes are now Interestingly, this location is also the site of insertion for the
called esc, and those type III secretion genes with no homologs retronphage fR73 and a large (70 kb) insert (PAI) of uro-
will continue to be called sep. Mutation of sepB (escN) abol- pathogenic E. coli strains containing genes for hemolysin (hly)
ishes secretion of EspA, EspB, and EspD and abolishes signal and P-related fimbriae (prf) (76). The large insert for uro-
transduction and the A/E phenotype (309). Secretion of the pathogenic E. coli has been termed a pathogenicity island (76),
110-kDa EspC protein is not abolished by mutation of sepB and the insertion of the EPEC LEE at the same site suggests
(escN) (309), and EspC apparently is secreted extracellularly that this region of the E. coli chromosome is a hot spot for
via a mechanism similar to that used by IgA protease of N. insertion of virulence factor genes. The G1C content of the
gonorrhoeae (617). Thus, EPEC possesses a specialized protein LEE is ca. 38%, which is strikingly lower than the 50 to 51%
secretion system that is necessary for translocation of critical G1C content of the total E. coli genome (431), thus suggesting
proteins from the bacterial cytoplasm to the external environ- horizontal transfer of this pathogenicity island into E. coli from
ment, where they can interact with epithelial cells. In Yersinia another species. The LEE pathogenicity island not only is
spp., the type III secretion system mediates the injection of the necessary for the A/E phenotype but also is sufficient. Mc-
Yop proteins directly into the eukaryotic cell (546), and a Daniel and Kaper (432) recently reported that a recombinant
similar mechanism could be true for EPEC, whereby the at- plasmid clone containing the entire LEE region with less than
tached EPEC bacterium directly injects one or more of the 800 bp of flanking DNA is sufficient to confer the A/E pheno-
EspA, EspB, and EspD proteins into the epithelial cell. type when cloned into K-12 or normal E. coli from the
Locus of enterocyte effacement. McDaniel et al. (431) have normal flora.
shown that the eae, espB, and sep (esc) genes are all located EAF plasmids. The BFP is encoded on plasmids which range
within a 35.5-kb chromosomal region of EPEC strain E2348/69 in size from ca. 50 to 70 MDa, called the EAF plasmids. These
(Fig. 8); subsequent studies showed that espA and espD but not plasmids share extensive homology among various EPEC
espC are also located in this region (352, 371, 617). This region, strains (467), and the restriction maps of two EAF plasmids
have been determined (467, 609). Downstream of the bfp gene B171 to HEp-2 cells by ca. 75% (243). Combining anti-FB171
cluster is a cluster of three genes encoding a transcriptional and anti-BFP sera inhibited localized adherence by ca. 100%.
activator (Per), which positively regulates several chromo- The need for multiple antisera to completely inhibit local-
somal and plasmid genes necessary for the pathogenesis of ized adherence and the results of ultrastructural studies of the
EPEC (see below). Beyond the per genes is a 1-kb restriction “microcolonies” comprising the localized adherence pheno-
fragment that has been extensively used as a diagnostic DNA type indicate that this phenomenon is multifactorial (243).
probe, called the EAF probe (27, 461). Although the DNA Scanning electron micrographs of the microcolonies reveal
sequence of this fragment has been determined (214), the that multiple bacterium–HEp-2 cell and bacterium-bacterium
contribution to EPEC pathogenesis of the genes encoded in interactions are involved. Thin fibers resembling fimbriae ap-
the EAF probe region is unknown, as is the contribution of pear to link bacterial cells and epithelial cells, while rope-like
genes contained in the major portion of the EAF plasmid. The structures resembling BFP may be primarily involved in bac-
use of the EAF probe for diagnosis of EPEC is discussed terium-bacterium interactions. Construction of isogenic mu-
below. tants specifically altered in genes encoding the various fimbrial
The importance of the EAF plasmid in human disease was structures of EPEC will be necessary before the localized ad-
shown by Levine et al. (386), who fed strain E2348/69 possess- herence phenotype is completely understood.
ing the EAF plasmid and a derivative of this strain that had lost (ii) EAST1. As described below, many EAEC strains pro-
the plasmid to adult volunteers. Diarrhea occurred in 9 of 10 duce a low-molecular-weight ST called EAST1. Some EPEC

volunteers who ingested the wild-type strain (mean diarrheal strains also produce EAST1, and a survey of E. coli strains by
stool volume, 1,178 ml) but in only 2 of 9 volunteers who Savarino et al. (564) reported that 14 of 65 EPEC strains tested
ingested the cured derivative (mean stool volume, of 433 ml) (22%) hybridized with the astA gene encoding EAST1. Inter-
(P , 0.006). Interestingly, although this plasmid is highly stable estingly, strain E2348/69, the prototype EPEC strain used for
in vitro (,1% spontaneous plasmid cure rate), 67% of the volunteer studies, contains two copies of the astA gene, one in
challenge strain isolates recovered from volunteer stool spec- the chromosome and one in the EAF plasmid. The significance
imens had lost this plasmid (386). This high rate of spontane- of this toxin in EPEC pathogenesis is unknown, but it is inter-
ous cure could affect the diagnosis of EPEC if possession of the esting that the EAF-negative EPEC-like organisms responsible
EAF plasmid is part of the definition (see below). for two large outbreaks of disease in adults in Minnesota and
Regulation. As for many other bacterial pathogens, expres- Finland (see below) also contained the astA gene. Determina-
sion of EPEC virulence factors is regulated by a trans-acting tion whether EPEC strains containing astA are more fre-
protein. A cluster of three open reading frames designated quently isolated from adults than are EPEC strains lacking
astA might yield insights into the striking age distribution seen
perA, perB, and perC (for plasmid-encoded regulator) encode
with infections due to EPEC (see below).
proteins that form a regulatory complex, which activates the
(iii) Invasion. Several investigators have shown that EPEC
transcription of several genes in the chromosome and on the
strains are capable of entering a variety of epithelial cell lines
EAF plasmid. The predicted protein product of the first open
(18, 156, 213, 448). Furthermore, many published photographs
reading frame of this region, PerA, has homology to the AraC
of animal and human EPEC infections show apparently intra-
family of bacterial regulators, including regulators of virulence
cellular bacteria (453, 524, 667, 669). However, unlike true
genes of Shigella spp. (VirF) and ETEC (Rns and CfaD/CfaR) intracellular pathogens such as Shigella spp., EPEC strains do
(257). The Per regulator increases expression of the chromo- not multiply intracellularly or escape from a phagocytic vacu-
somal eae (257) and espB (eaeB) genes (255), as well as that of ole and thus do not appear to be specifically adapted for
genes encoding 50- and 33-kDa OMPs (257). Secretion of intracellular survival. EPEC strains do not cause dysentery or
EspB is regulated by Per and is induced in response to condi- a typhoid-like syndrome, and so the clinical significance of cell
tions similar to those in the gastrointestinal tract (349). The entry in the pathogenesis of disease due to EPEC is not clear.
bfpA gene is also under per control, as the recently described However, despite the lack of evidence that invasion by EPEC
bfpTVW regulatory gene cluster is in fact allelic with perABC is important in pathogenesis, this phenotype has been very
(256, 329, 651). In addition to increasing the expression of useful in studying the molecular genetics of EPEC because
these genes, Per decreases the expression of a gene encoding a many of the genes involved in invasion are also involved in
20-kDa OMP (257). It has also been reported that Per can forming the A/E lesion. Donnenberg et al. (155) have used
repress the expression of intimin during the stationary phase of TnphoA and the gentamicin protection assay to isolate mutants
growth while activating the expression of intimin during expo- deficient in cell entry. Two categories of noninvasive mutants
nential growth (357). Thus, there appears to be a global reg- had insertions located in the bfpA or dsbA loci, both of which
ulatory system for regulation of virulence in EPEC that allows are required to produce functional BFP fimbriae (157, 714). A
this pathogen to respond to different environmental conditions third category of mutants were deficient in intimate adherence,
and different phases of growth, but the details of this regula- and these mutants had insertions in the eae and espB (eaeB)
tion have not been elucidated. genes (155, 164). Another category of mutants (category 4
Other potential virulence factors. (i) Other fimbriae. There mutants or cfm) were completely deficient in the FAS test and
have been numerous reports of additional fimbrial structures in the ability to induce a tyrosine kinase activity in the host cell;
produced by EPEC, some of which were subsequently shown the mutations in these mutants were subsequently shown to be
to be type 1 fimbriae (reviewed in reference 376). Girón et al. in the sep/esc genes encoding the type III protein secretion
(243) reported an extensive characterization of fimbriae pro- system. These results indicate that there is significant overlap
duced by EPEC strain B171. In addition to BFP, this strain between the genes responsible for the invasion process and
produced rod-like fimbriae and fibrillae with subunit sizes of genes involved in producing attaching and effacing lesions.
16.5, 15.5, and 14.7 kDa. Their N-terminal amino acid se- Two studies have reported that O111:NM strains contain
quence showed homology to F9 and F72 fimbriae of uropatho- plasmid sequences that can confer invasiveness upon E. coli
genic E. coli (both are P fimbriae) and the F1845 fimbria of K-12 strains containing the cloned fragments (208, 567). Se-
DAEC, respectively. Antisera prepared against a mixture of all quences homologous to these cloned genes were present in
three fimbriae (called FB171) reduced the adherence of strain only a minority of EPEC strains and were apparently not
contained in the LEE pathogenicity island. Mutation of the (108 to 1010) are given after gastric acid is neutralized with
sepZ locus within the LEE abolished the invasion phenotype bicarbonate (382). The infectious dose in naturally transmitted
without affecting the A/E phenotype (527). The relevance of infection among infants is not known but is presumed to be
these sequences to the pathogenesis of disease is unknown. much lower. The reason(s) for the relative resistance of adults
Mechanism of diarrhea. The impressive advances in our and older children is not known, but loss of specific receptors
understanding of EPEC pathogenesis at the genetic and cel- with age is one possibility. A similar restriction of disease to
lular levels allow us to present a plausible mechanism for how young animals is seen with E. coli strains that cause diarrhea by
diarrhea results from infection with EPEC. The dramatic loss similar A/E mechanisms in weanling rabbits. The fact that
of the absorptive microvilli in the A/E lesion could lead to EPEC has not been implicated as a cause of traveler’s diarrhea
diarrhea via malabsorption. However, the incubation period in in countries with high incidences of both EPEC and ETEC
adult volunteers can be very short: as little as 2.9 h between suggests a physiological basis for this resistance rather than
ingestion of the organisms and the onset of diarrhea (161). host immunity or exposure. However, several outbreaks of
This rapidity suggests that a more active secretory mechanism diarrhea due to EPEC have been reported in healthy adults
is involved in diarrhea caused by EPEC and that a variety of (135, 281, 580, 681), presumably due to ingestion of a large
intracellular mediators of intestinal ion transport, such as cal- inoculum from a common source. Sporadic disease has also
cium, PKC, inositol phosphates, and tyrosine kinase, are af- been seen in some adults with compromising factors (diabetics,
fected by EPEC infection. There have been recent reports of those with achlorhydria, the elderly) (384, 620).

EPEC actively altering ion transport in epithelial cells. Stein et Transmission and reservoirs. As with other diarrheagenic E.
al. (618) found a significant decrease in the transmembrane coli strains, transmission of EPEC is fecal-oral, with contami-
potential in epithelial cells infected with EPEC. This result nated hands, contaminated weaning foods or formula, or con-
suggests that EPEC stimulates either an influx of positive ions taminated fomites serving as vehicles (384). Unless strict de-
or an efflux of negative ions across the membrane. Knutton et contamination procedures are followed, admission of an infant
al. (361) have found that EPEC can stimulate a rapid but to a pediatric ward can result in contamination of crib linen,
transient increase in short-circuit current (Isc) in intestinal toys, tabletops, hand towels, scales, carriages, rubber nipples,
epithelial cells mounted in Ussing chambers; secretion of chlo- etc. In one study (543), EPEC was isolated from dust and
ride ions was implicated in this effect (132). In both studies, aerosols, suggesting potential airborne transmission, either di-
changes in ion transport did not occur as a result of addition of rectly through inhalation followed by ingestion or indirectly via
cell-free culture supernatants of EPEC to epithelial cells; in- contamination of other fomites. In the uncommon adult out-
fection with viable bacteria was necessary. These ionic changes breaks, waterborne and foodborne transmission has been re-
were abrogated by mutation of the espB gene but not the eae ported but no particular type of food has been implicated as
gene (361, 618). more likely to serve as a source of infection. The reservoir of
Mechanisms other than direct stimulation of ion secretion EPEC infection is thought to be symptomatic or asymptomatic
are also possible. As described above, infection of an epithelial children and asymptomatic adult carriers, including mothers
cell monolayer by EPEC leads to decreased monolayer resis- and persons who handle infants (384). Numerous studies have
tance, which could result in diarrhea due to increased intestinal documented the spread of infection through hospitals, nurser-
permeability. Some clinical reports have noted a local inflam- ies, and day care centers from an index case (82, 384, 703).
matory response upon biopsy (669), and inflammation has Epidemiologic studies in several countries have shown high
been seen in animal models of EPEC infection (662). As noted backgrounds of asymptomatic carriage; in some studies, as
above, attachment of EPEC to cultured human intestinal epi- many as 17 to 20% of healthy infants younger than 2 years shed
thelial monolayers induces the transmigration of PMNs (565). E. coli of EPEC serotypes in their stools (reviewed in reference
Transmigration of PMNs can, in turn, result in increased Isc 384). In symptomatic patients, EPEC can be isolated from
due to chloride secretion (403), thereby suggesting another stools up to 2 weeks after cessation of symptoms (289). Al-
mechanism of diarrhea due to EPEC. though animals such as rabbits, pigs (717), and dogs have
These multiple mechanisms could all be involved in diarrhea EPEC-like organisms associated with disease, the serotypes
due to EPEC. The active chloride secretion, perhaps mediated found in these animal pathogens are usually not human sero-
via a pathway involving PKC, could account for the rapid onset types.
of diarrhea. The prolonged diarrhea seen in some patients EPEC in developed countries. EPEC once caused frequent
could result from malabsorption due to loss of the brush bor- outbreaks of infant diarrhea in the United States and the
der. A local inflammatory response and increased intestinal United Kingdom (541). These community-acquired and noso-
permeability in response to EPEC infection could also contrib- comial outbreaks were often explosive, with up to 50% mor-
ute to the diarrhea. tality (239, 384, 541). EPEC strains are no longer as important
a cause of diarrhea in developed countries as they were in the
Epidemiology 1940s and 1950s; the reasons for this change in incidence are
not clear. The discontinuation of routine serological screening
Age distribution. The most notable feature of the epidemi- of E. coli by most hospitals contributes in part to the low
ology of disease due to EPEC is the striking age distribution incidence of EPEC reported in developed countries. However,
seen in persons infected with this pathogen. EPEC infection is several outbreaks of diarrhea due to EPEC have been reported
primarily a disease of infants younger than 2 years. As re- in the last two decades in the United States, the United King-
viewed by Levine and Edelman (384), numerous case-control dom, Finland, and other developed countries. These outbreaks
studies in many countries have shown a strong correlation of frequently occur in day care centers (82, 509) and occasionally
isolation of EPEC from infants with diarrhea compared to occur in pediatric wards (82, 547). An outbreak due to atypical
healthy infants. The correlation is strongest with infants EPEC was recently reported among adults who ate at a gour-
younger than 6 months. In children older than 2 years, EPEC met buffet in Minnesota (see below) (281). However, EPEC
can be isolated from healthy and sick individuals, but a statis- strains are also associated with sporadic cases of diarrhea in
tically significant correlation with disease is usually not found. the United States and other developed countries (78, 384, 592).
EPEC can cause diarrhea in adult volunteers if high inocula A recent study of E. coli isolated from children with diarrhea in
Seattle used diagnostic DNA probes and found a high inci- Other therapies such as bismuth subsalicylate (204) and spe-
dence of EPEC-like organisms in this population (78). This cific bovine anti-EPEC milk Igs (447) have also proven useful.
study found that eae-positive, stx-negative strains (usually lo- There are no vaccines currently available or in clinical trials to
calized adherence positive but EAF negative) were isolated prevent disease due to EPEC.
from 3.6% of specimens, a frequency that exceeded the rates of
recovery of Campylobacter spp., E. coli O157:H7, Salmonella Detection and Diagnosis
spp., Shigella spp., or Yersinia spp. These results suggest that
the current importance of EPEC in the United States may be Definition of EPEC. Before discussing the detection and
seriously underestimated. diagnosis of EPEC, it is appropriate to first discuss the char-
EPEC in developing countries. In contrast to the limited acteristics that actually define EPEC. The definition of EPEC
importance of EPEC in developed countries, EPEC is a major has changed drastically in recent years as our knowledge of this
cause of infant diarrhea in developing countries. Numerous pathogen has grown. For many years these organisms were
case-control studies on six continents have found EPEC to be defined only by O serogroups, which were subsequently refined
more frequently isolated from infants with diarrhea than from to O:H serotypes. This definition changed as additional sero-
matched healthy controls (reviewed in references 154 and 384). types were associated with infantile diarrhea (184). Citing re-
Particularly in the 0- to 6-month age group, EPEC strains are cent pathogenesis data, the Second International Symposium
often the most frequently isolated bacterial diarrheal patho- on EPEC in 1995 reached a consensus on the basic character-

gens. Studies in Brazil (251, 252, 653), Mexico (140, 141), and istics of EPEC (328); the most important of these were the A/E
South Africa (542) have shown that 30 to 40% of infant diar- histopathology and the absence of Shiga toxin. Many EHEC
rhea can be attributed to EPEC, and in some studies EPEC strains also produce the A/E lesion; therefore, determining the
infection exceeds rotavirus infection in incidence (140, 251, presence (indicative of EHEC) or absence (indicative of
252, 540). EPEC strains are an important cause of disease in all EPEC) of Stx is essential. The possession of specific O and H
settings—nosocomial outbreaks, outpatient clinics, patients antigens, which for many years was the sole defining microbi-
admitted to hospitals, community-based longitudinal studies, ological characteristic of EPEC, is no longer deemed an es-
and urban and rural settings. Several studies have shown that sential characteristic of EPEC, although the majority of EPEC
breast feeding is protective against diarrhea due to EPEC (74, strains fall into certain well-recognized O:H serotypes.
540). Both human colostrum and milk strongly inhibit the There is some debate whether EPEC strains that lack the
adhesion of EPEC to HEp-2 cells in vitro, and the inhibitory EAF plasmid (which encodes the BFP adhesin and Per regu-
activity has been found in both sIgA and oligosaccharide frac- lators) are true pathogens (328). In every case-control study of
tions (100, 143). FAS-positive, eae-positive EPEC strains so far reported, only
EAF-positive strains and not EAF-negative strains were sig-
Clinical Considerations nificantly associated with diarrhea (183, 252, 254, 387). EAF-
negative strains that produce the A/E lesion (i.e., are FAS
EPEC causes primarily acute diarrhea, although many cases positive or eae probe positive) can result from loss of the EAF
of protracted EPEC diarrhea have also been reported (154, plasmid; indeed, it has been well documented that volunteers
384). In the Cincinnati outbreak reported by Rothbaum et al. who ingest an EAF-positive strain can shed derivatives of the
(547), the duration of hospitalization ranged from 21 to 120 challenge strain that have spontaneously lost the EAF plasmid
days. The infection can often be quite severe, and many clinical (386). Furthermore, EAF-negative strains isolated during the
reports emphasize the severity of the disease (82, 547). In course of an epidemiological study could also be derivatives of
outbreaks in the United States and the United Kingdom in the EHEC that have lost the phages that encode Stx. At present, it
mid-20th century, mortality rates of 25 to 50% were reported is impossible to distinguish strains of these two categories from
(154), and in recent outbreaks from developing countries, 30% strains that never possessed the EAF plasmid but can none-
mortality was reported (590). In developed countries with the theless cause diarrhea in certain individuals. Moreover, volun-
full range of modern treatment available, mortality is much teer studies have shown that an EAF plasmid-cured derivative
lower although deaths can still result (547). In addition to of E2348/69, while significantly attenuated relative to the EAF-
profuse watery diarrhea, vomiting and low-grade fever are positive parent strain, nonetheless caused mild diarrhea in two
common symptoms of EPEC infection. Fecal leukocytes are of nine volunteers (386). EAF-negative, eae-positive strains
seen only occasionally, but more sensitive tests for inflamma- have been implicated as the causative agents in at least one
tory diarrhea such as an anti-lactoferrin latex bead agglutina- small pediatric outbreak in the United Kingdom (300, 602) and
tion test are frequently positive with EPEC infection (449). one large outbreak involving more than 600 individuals in
Proximal small intestinal mucosal biopsy specimens often, but Finland (602, 681). However, the latter outbreak was highly
not always, show intimately adherent bacteria and the classic unusual in that a number of adults were affected. In a survey of
A/E histopathology (194, 547, 592). The presence of the A/E 925 E. coli strains isolated from patients with diarrhea in the
lesion is associated with disarrangement of the digestive-ab- United Kingdom and belonging to EPEC serogroups, fewer
sorptive enzyme system, leading to malabsorption of nutrients than 10% of eae-positive strains hybridized with the EAF
(194, 289, 547). probe (585). EAF-negative EPEC strains may be similar to
As with other diarrheal pathogens, the primary goal of treat- LT-only ETEC strains, which are usually not associated with
ment of EPEC diarrhea is to prevent dehydration by correcting diarrhea in case-control studies but which have been impli-
fluid and electrolyte imbalances. Oral rehydration may be suf- cated in outbreaks and sporadic cases. Whether EAF-negative
ficient for milder cases, but more severe cases require paren- strains possess additional virulence factors that have yet to be
teral rehydration. Correction of nutritional imbalance with lac- discovered or whether there are specific host factors that pre-
tose-free formula or breast milk may be insufficient for some dispose to disease with these strains is unknown.
severely ill patients, and total parenteral nutrition may be re- Accordingly, a consensus definition was achieved at the Sec-
quired (154, 194). A variety of antibiotics have been used to ond International Symposium on EPEC: A/E, Stx-negative
treat EPEC and have proved useful in many cases (154), but strains possessing the EAF plasmid would be called “typical
multiple antibiotic resistance is common for EPEC (154). EPEC,” while such strains that do not possess the EAF plas-
mid would be called “atypical EPEC.” Several outbreaks have (a) eae gene. Possession of eae sequences correlates with
implicated atypical EPEC as the causative agent. One recent possession of the 35-kb LEE pathogenicity island encoding
foodborne outbreak involved more than 100 adults who ate at A/E (431). No exceptions to this generalization have been
a gourmet buffet in Minnesota (281). The implicated organism reported, and so there is no need to test for other sequences in
was an O39:NM E. coli strain that hybridized with the eae gene the LEE such as espB and sep unless specific strain differences
plus other genes in the LEE but did not hybridize with the are sought for molecular epidemiology purposes. Sequence
EAF probe. In addition, this strain hybridized with a probe variability has been reported for the espB sequences from dif-
(astA) to EAEC EAST1. In an outbreak of diarrhea in Finnish ferent EPEC and EHEC strains (182). As discussed above,
adults and schoolchildren, an O111 eae-positive, EAF-nega- sequence variability is also seen in the 39 end of the eae gene
tive, astA-positive E. coli strain was implicated (602, 681). Un- encoding the C-terminal region of intimin. A 1-kb fragment
like the O39:NM strain, the O111 strain possessed a standard probe originally described by Jerse et al. (314) is derived from
EPEC O antigen and also exhibited localized adherence on sequences encoding the highly conserved N-terminal region.
tissue cells in culture, although it was EAF negative. Note that Compared to the A/E phenotype as determined by the FAS
the Vi antigen reportedly expressed by this strain could not be test, this probe was 100% sensitive and 98% specific; the two
confirmed in reference laboratories (602). “false-positive” strains possessed the LEE but were FAS neg-
Diagnostic tests. Given that EPEC strains, as with other ative due to reduced expression of the genes on the LEE (311,
diarrheagenic E. coli strains, are defined on the basis of viru- 314). This probe is easily used with nonradioactive labeling

lence properties, there are two approaches to the detection of methods (463). Fragment probes using the variable 39 end of
EPEC in the laboratory: phenotypic and genotypic. The phe- the eae gene have been reported for O157:H7 EHEC strains
notypic approach requires the use of cell cultures and fluores- (691), but no probes can successfully differentiate all EPEC eae
cence microscopy, and the genotypic method requires the use sequences from all EHEC eae sequences.
of DNA hybridization or PCR. A variety of eae primers for detecting this gene by PCR have
(i) Phenotypic tests. The A/E phenotype can be identified by been tested (eae PCR is discussed in the section on EHEC,
using cultured HEp-2 or HeLa cells and the mushroom toxin below). As reported by Gannon et al. (234), primer pairs for
phalloidin conjugated to FITC or rhodamine (available from the conserved 59 region amplified fragments from all eae-pos-
Sigma, Molecular Probes, and other sources) as in the FAS itive strains whereas primers from the 39 end were specific for
assay originally described by Knutton et al. (359). In brief, the certain serotypes. Primer sequences which will detect eae in
bacterial cells are incubated with the cultured cells for 3 to 6 h, both EPEC and EHEC strains are shown in Table 2.
after which cells are fixed and washed as described above for (b) EAF plasmid. The 1-kb EAF fragment probe originally
the HEp-2 adherence assay. The cells are permeabilized by described by Nataro et al. (461) is from a region of the EAF
treating coverslips with 0.1% Triton X-100 in phosphate-buff- plasmid with an unknown function. This probe may be used
ered saline (pH 7.2) for 5 min, followed by subjecting them to with nonradioactive labeling techniques (238). A sensitive and
multiple washes in phosphate-buffered saline and treating specific oligonucleotide probe consisting of 21 bases of the
them with conjugated phalloidin to stain the filamentous actin. EAF fragment was developed by Jerse et al. (313). Franke et
The specimens are then examined under an incident-light flu- al. (214) developed a PCR primer pair to amplify a 397-bp
orescence microscope (at the proper wavelength for FITC or region of the EAF probe. The sequences of these probes and
rhodamine conjugate), and the same fields are visualized under primers are given in Table 2.
phase-contrast microscopy. Punctate spots of intense fluores- The cloning of the bfpA gene allowed the development of
cence that correspond to bacterial cells under phase-contrast DNA probes for specific plasmid-encoded virulence factors.
microscopy denote a positive FAS test. To avoid the use of the Girón et al. (241) reported an 850-bp bfpA fragment probe that
highly toxic phalloidin, Ismali et al. (303) used a monoclonal was slightly more sensitive than the EAF probe; among EPEC
antibody directed against a-actinin, which is also concentrated strains exhibiting localized adherence to HEp-2 cells, the bfpA
in epithelial cells directly beneath the adherent EPEC. The and EAF probes hybridized with 99 and 96% of the strains,
A/E phenotype can also be identified by electron microscopic respectively. Trabulsi et al. (658) have reported that bfpA-
examination of intestinal biopsy specimens or of cultured ep- positive, EAF-negative EPEC strains are found in specific se-
ithelial cells incubated with EPEC. rotypes such as O119:H2. Sohel et al. (608) used a 579-bp bfpA
There are a number of phenotypic tests to determine the probe cloned from a different EPEC strain which hybridized to
other crucial characteristic of EPEC, the lack of Stx expression. a number of Salmonella strains under moderate- but not high-
These tests are discussed in the section on EHEC below. An stringency conditions. A 29-base oligonucleotide probe for the
excellent phenotypic marker for the presence of the EAF bfpA gene was reported by Nagayama et al. (458). This non-
plasmid is localized adherence on HEp-2 or HeLa cells, as radioactive probe, conjugated to alkaline phosphatase, had a
described above. An ELISA for the detection of EAF-positive sensitivity and specificity of 95.7 and 100%, respectively, with
EPEC, based on an antiserum raised against an EAF plasmid- respect to assays for localized adherence. Gunzburg et al. (268)
containing strain and absorbed against a plasmid-cured strain, reported a highly sensitive and specific PCR procedure to
has been described previously (10). However, no subse- detect the bfpA gene, and this technique was readily adapted
quent reports have appeared in which this test was described. for the diagnosis of EPEC in a field setting in Northern Brazil
There are no readily available immunoassays to detect the (654). The sequences for the bfpA oligonucleotide probes and
A/E pattern. PCR primers are given in Table 2.
(ii) Genotypic tests. DNA probes and PCR primers have In general, there is excellent agreement among the pheno-
been developed and evaluated for the three major character- typic characteristics of localized adherence on HEp-2 cells, the
istics of EPEC: A/E, EAF plasmid, and lack of Shiga toxin. presence of bfpA sequences, and EAF probe positivity. How-
DNA tests for genes encoding Shiga toxin are reviewed below. ever, there are reports of an LA pattern expressed by EPEC
As defined above, typical EPEC strains would possess the eae strains that are EAF probe negative (78, 586). Whether these
gene for A/E and the EAF probe or bfp sequences, indicating strains express another adhesin analogous to BFP is not
the presence of the EAF plasmid. Atypical EPEC strains known, but it has also been shown that increased expression of
would possess the eae gene only without the EAF plasmid. the chromosomal eae gene encoding intimin in the absence of
the EAF plasmid can lead to an LA-like adherence pattern on spp., or is truly a novel pathogen that has recently arisen. After
HEp-2 cells (259). Variability in cell culture assays could also E. coli O157:H7 was recognized as a cause of HC, the Centers
account for some of these discrepancies. The EAF probe has for Disease Control and Prevention (CDC) reviewed over
been used in dozens of epidemiologic studies all over the 3,000 E. coli strains serotyped between 1973 and 1983 and
world, and there are several studies showing the significant found only 1 O157:H7 isolate (539). The Public Health Labo-
correlation of possession of EAF probe sequences and disease ratory in the United Kingdom also found only 1 O157:H7
potential. In view of the many years of data that have accumu- strain among 15,000 E. coli strains serotyped between 1978 and
lated with the EAF probe, we favor the continued use of the 1982, and the Laboratory Centre for Disease Control in Can-
EAF probe so that comparisons to previous studies can be ada found 6 O157:H7 strains among 2,000 isolates from pa-
readily made. However, the absence of EAF probe sequences tients with diarrhea between 1978 and 1982 (148, 263, 317).
in eae-positive strains that lack stx would still allow a diagnosis Therefore, it appears that the presence of O157:H7 strains has
of atypical EPEC under the definition discussed above. genuinely increased in recent years and was not simply missed
prior to 1982. However, HUS was a well-known clinical entity
ENTEROHEMORRHAGIC E. COLI prior to 1982. Since its initial description in 1955, numerous
outbreaks of HUS gave credence to the hypothesis that HUS
was due to a bacterial or viral agent (338). Although Stx-
Origins
producing S. dysenteriae 1 strains were clearly associated with

The recognition of EHEC as a distinct class of pathogenic E. HUS, stool cultures obtained during many HUS outbreaks
coli resulted from two key epidemiologic observations. The yielded E. coli but no recognized pathogens. In a remarkably
first was the 1983 report by Riley et al. (539), who investigated prescient 1968 article describing HUS in South Africa, Kibel
two outbreaks of a distinctive gastrointestinal illness charac- and Barnard (355) suggested that a “mutant strain of E. coli”
terized by severe crampy abdominal pain, watery diarrhea fol- mutated by a bacteriophage may be responsible for this syn-
lowed by grossly bloody diarrhea, and little or no fever. This drome. In the 1980s, it was recognized that the Stx is encoded
illness, designated hemorrhagic colitis (HC), was associated on a bacteriophage in E. coli (484, 587, 605) and that over 100
with the ingestion of undercooked hamburgers at a fast-food different E. coli serotypes can express Stx (338). O157:H7
restaurant chain. Stool cultures from these patients yielded a strains are closely related to Stx-negative O55:H7 EPEC
previously rarely isolated E. coli serotype, O157:H7. The sec- strains (690), a serotype that has long been associated with
ond key observation was by Karmali et al. (343), also in 1983, worldwide outbreaks of infant diarrhea, and EHEC and EPEC
who reported the association of sporadic cases of hemolytic share many intestinal adherence and other virulence factors
uremic syndrome (HUS) with fecal cytotoxin and cytotoxin- (see below). Thus, it appears that non-O157:H7 E. coli strains
producing E. coli in stools. HUS (defined by the triad of acute producing Stx have been around for several decades but it was
renal failure, thrombocytopenia, and microangiopathic hemo- only with the emergence in the early 1980s of the O157:H7
lytic anemia) was already known to be preceded typically by a clone that this pathogenic class of E. coli was recognized.
bloody diarrheal illness indistinguishable from HC. Thus, two The discovery of this pathogen along parallel paths of inves-
key clinical microbiological observations, one based on a rare tigation resulted in a parallel nomenclature system, a situation
E. coli serotype and the other based on production of a specific that still exists. The term “verotoxigenic E. coli” or “Vero
cytotoxin, led to the recognition of a novel and increasingly cytotoxin-producing E. coli” (VTEC) was derived from the
important class of enteric pathogens causing intestinal and 1977 observation by Konowalchuk et al. (366) that these strains
renal disease. produced a toxin that was cytotoxic for Vero cells. An alter-
The cytotoxin assay used by Karmali et al. (343) was origi- native nomenclature is “Shiga toxin-producing E. coli” (STEC
nally reported by Konowalchuk and colleagues in 1977 (366). [formerly SLTEC]), which reflects the fact that one of the
These investigators reported that culture filtrates from some cytotoxins produced by these organisms is essentially identical
strains of E. coli produced a striking, irreversible cytopathic at the genetic and protein levels to the Stx produced by S.
effect on cultured Vero cells that was quite distinct from the dysenteriae I; the discovery of Stx nearly 100 years ago far
noncytopathic effect of ETEC LT on CHO or Y-1 cells. At this pre-dates the description by Konowalchuk. The arguments for
same time, O’Brien et al. reported (481, 486) that extracts of and against each term have been published by their proponents
certain E. coli strains were cytotoxic for HeLa cells and that (99, 339) and will not be repeated here; usage within the
this cytotoxic activity could be neutralized by antitoxin pre- scientific community will ultimately determine whether one or
pared against crude Shigella dysenteriae 1 (Shiga) toxin (Stx). both names will be used. STEC and VTEC are equivalent
They subsequently reported that many E. coli strains isolated terms, and both refer to E. coli strains that produce one or
from diarrheal illness produced a Shiga-like toxin (SLT), in- more toxins of the Stx family (see below). However, it is not
cluding one of the strains reported by Konowalchuk et al. (366) clear that mere possession of genes encoding Stx confer patho-
to produce the Vero cytotoxin (482). O’Brien et al. subse- genicity in the absence of other virulence factors (described
quently showed (483) that Shiga-like toxin and the Vero cyto- below). The term “enterohemorrhagic E. coli” (EHEC) was
toxin were the same toxin and that the O157:H7 strains de- originally coined to denote strains that cause HC and HUS,
scribed by Riley et al. produced this toxin. Independently, express Stx, cause A/E lesions on epithelial cells, and possess a
Johnson et al. (317) reported that E. coli O157:H7 strains ca. 60-MDa plasmid (381, 384). Thus, EHEC denotes a subset
isolated from patients with HC in Canada produced a cytotoxin of STEC and includes a clinical connotation that is not implied
active on Vero cells. Karmali et al. (341) concluded an eventful with STEC. Whereas not all STEC strains are believed to be
publication year for this pathogen by proposing that Vero pathogens, all EHEC strains by the above definition are con-
cytotoxin/Shiga-like toxin was the common virulence factor sidered to be pathogens. For this review, we will use the term
between HC and HUS and was responsible for damage to both “typical EHEC” to denote STEC strains such as O157:H7 that
intestinal and renal tissue. produce Stx and A/E lesions and possess the 60-MDa plasmid
As with any organism that is termed an “emerging patho- and the term “atypical EHEC” to denote STEC strains that do
gen,” the question arises whether the organism has always not produce A/E lesions and/or do not possess the ca. 60-MDa
been present and was simply undiagnosed, as with Legionella EHEC plasmid.
Pathogenesis Shiga toxins. The major virulence factor, and a defining

characteristic of EHEC, is Stx. This potent cytotoxin is the
Most of the work on pathogenic factors of E. coli O157:H7 factor that leads to death and many other symptoms in patients
has focused on the Stx, which are encoded on a bacteriophage infected with EHEC. The Stx family has been extensively re-
inserted into the chromosome. Additional potential virulence viewed (479, 480, 485, 589, 645), and additional primary ref-
factors are encoded in the chromosome and on a ca. 60-MDa erences will be found in these reviews.
plasmid found in all EHEC strains of serotype O157:H7. (i) Structure and genetics. The Stx family contains two ma-
Histopathology. The classic intestinal histopathology char- jor, immunologically non-cross-reactive groups called Stx1 and
acteristic of E. coli O157:H7 infection includes hemorrhage Stx2. A single EHEC strain may express Stx1 only, Stx2 only, or
and edema in the lamina propria (262). The edema and sub- both toxins or even multiple forms of Stx2. Stx1 from EHEC is
mucosal hemorrhage in the ascending and transverse colon can identical to Shiga toxin from S. dysenteriae I. (Stx1 from some
be manifested as a “thumbprinting” pattern on barium enema strains may differ from Stx in one residue, while Stx1 from
radiography (84, 539). Colonic biopsy specimens from many other strains shows no sequence variation [485, 632].) The
patients also show focal necrosis and infiltration of neutrophils. prototypical Stx1 and Stx2 toxins have 55 and 57% sequence
The overall pattern resembles a combination of ischemic and identity in the A and B subunits, respectively (306). While Stx1
infectious injuries similar to those described in toxin-mediated is highly conserved, sequence variation exists within Stx2. The
different variants are designated Stx2c, Stx2v, Stx2vhb, Stx2e,

Clostridium difficile-associated colitis, and pseudomembranes
etc., and the various subtypes are wholly interchangeable be-
are seen in many patients (262).
tween the Stx and VT nomenclatures (i.e., Stx1 5 VT1,
The classic A/E histopathology has been seen in gnotobiotic
Stx2e 5 VT2e, etc.) (99).
piglets (162, 434, 662, 663), infant rabbits (498), and cultured
The basic A-B subunit structure is conserved across all mem-
epithelial cells (302, 359) infected with E. coli O157:H7. How-
bers of the Shiga toxin family. For the prototype toxin of this
ever, A/E lesions have not yet been reported from clinical
family, Shiga toxin, the single 32-kDa A subunit is proteolyti-
specimens. The failure to detect A/E lesions in clinical speci-
cally nicked to yield a ca. 28-kDa peptide (A1) and a 4-kDa
mens is thought to be because colonic biopsy specimens are
peptide (A2); these peptides remain linked by a disulfide bond.
usually collected late in the disease and A/E lesions would be
The A1 peptide contains the enzymatic activity, and the A2
present only early in the course, before the potent cytotoxic peptide serves to bind the A subunit to a pentamer of five
effect of Stx occurs (636). In addition, the fear of gastrointes- identical 7.7-kDa B subunits. The B pentamer binds the toxin
tinal hemorrhage during HUS and HC raises the clinical to a specific glycolipid receptor, globotriaosylceramide or Gb3,
threshold for performing intestinal biopsy. which is present on the surface of eukaryotic cells. While Gb3
The cellular responses leading to the A/E histopathology is the main receptor for Stx, the Stx2e variant uses Gb4 as the
due to EHEC have not been studied as thoroughly as they have receptor. Stx2e is classically associated with pig edema disease
been with EPEC. High concentrations of polymerized actin are rather than human disease, but occasional strains that express
seen in EHEC mucosal lesions (359), and increased levels of only this variant are isolated from patients with HUS or diar-
IP3 and intracellular calcium are observed (302). However, in rhea (519, 647). After binding, the holotoxin is endocytosed
contrast to EPEC strains, EHEC strains fail to induce tyrosine through coated pits and is transported to the Golgi apparatus
phosphorylation of epithelial cell proteins (302). Interestingly, and then to the endoplasmic reticulum (reviewed in reference
an eae-negative EHEC strain (that presumably lacked the en- 556). The A subunit is translocated to the cytoplasm, where it
tire LEE) was still capable of increasing intracellular calcium acts on the 60S ribosomal subunit. Specifically, the A1 peptide
levels even though A/E lesions were not seen. These results is an N-glycosidase that removes a single adenine residue from
suggest that there are some differences between the cellular the 28S rRNA of eukaryotic ribosomes, thereby inhibiting pro-
response to EPEC and the response to EHEC (302). tein synthesis. The resulting disruption of protein synthesis
As described above for EPEC, the 35-kb LEE pathogenicity leads to the death of renal endothelial cells, intestinal epithe-
island which confers the A/E phenotype for EPEC is also lial cells, Vero or HeLa cells, or any cells which possess the
present in E. coli O157:H7. Genes involved in EHEC patho- Gb3 (or Gb4 for Stx2e) receptor.
genesis are illustrated in Fig. 9. The EHEC LEE contains The structural genes for Stx1 and Stx2 are found on lyso-
genes encoding intimin, the secreted proteins EspA and EspB, genic lambdoid bacteriophages; the genes for Stx2 are chro-
and a type III secretion pathway (310, 431). Interestingly, the mosomally encoded. (The stxAB genes encoding the A and B
sequences of espB genes from two different serotypes of EHEC subunits are also called sltAB and vtxAB in the literature.)
have only 80% homology at the predicted protein level (182). Production of Stx1 from E. coli and S. dysenteriae is repressed
Whether this sequence divergence in a key effector molecule by iron and reduced temperature, but expression of Stx2 is
has any effect on the pathogenesis of disease is not known. unaffected by these factors. During the early 1980s, there were
Similar to EPEC, E. coli O157:H7 induces a host inflamma- reports of production of low levels of Stx (SLT) by other
tory response that is apparently linked to the A/E histopathol- species such as V. cholerae, V. parahaemolyticus, Campylobacter
ogy. In a rabbit model, PMN infiltration in response to E. coli jejuni, E. coli K-12, and E. coli strains from the normal flora
O157:H7 infection was blocked by the addition of anti-CD18 (479). The stx genes are not present in these other species, and
antibody (187). Inhibition of this inflammatory response led to other investigators have been unable to confirm these initial
reduction but not elimination of diarrhea in this model. In- observations (94). However, some strains of Citrobacter freun-
creased levels of IL-8 have also been reported in cultured dii and Enterobacter spp. produce an Stx2 toxin and contain an
epithelial cells infected with EHEC (323). Isogenic E. coli stx2 gene with high homology to those found in E. coli (577).
O157:H7 mutants deficient in secretion of EspA or EspB have (ii) Stx in intestinal disease. There is a variety of data
not been tested in these systems, but based on results with showing the involvement of Stx in diarrhea and enterocolitis,
EPEC (565), it is likely that mutation of the genes encoding beginning with early demonstrations that purified Stx can cause
these factors would eliminate this inflammation. It should be fluid accumulation and histological damage when injected into
noted, however, that PMN are present in the stools of fewer ligated intestinal loops (reviewed in reference 479). One pos-
than 50% of patients with HC (261). sible mechanism for fluid secretion in response to Stx involves

FIG. 9. Genes involved in EHEC pathogenesis. Genes involved in EHEC pathogenesis are similar to those implicated for EPEC, except for the presence of the
Stx-encoding phage on the EHEC chromosome and the presence of the characteristic EHEC 60-MDa plasmid instead of the EAF plasmid of EPEC. The EHEC
plasmid is known to encode the enterohemolysin (ehx) as well as a fimbrial antigen potentially involved in colonization.
the selective killing of absorptive villus tip intestinal epithelial rious histological lesions including vascular changes, edema,
cells by Stx (325, 346). In rabbit ileum, the Gb3 receptor is and more severe inflammation. Similarly, Fontaine et al. (210)
present in much higher concentrations in villus cells than in the fed a strain of S. dysenteriae I specifically mutated in stx to
secretory crypt cells, and so the death of absorptive cells and monkeys and found that the disease was less severe in monkeys
preservation of secretory crypt cells could shift the usual bal- fed the Stx-negative mutant than in those fed the Stx-positive
ance of intestinal absorption and secretion toward net secre- parent strain. Infections with the two strains resulted in equiv-
tion (325). The available evidence therefore suggests that un- alent diarrheal stool volumes, but in animals receiving the
like LT or CT, Stx does not increase active secretion of Cl2 Stx-positive strain, the stools were consistently more bloody
ions. Intravenous administration of purified Stx1 or Stx2 to and there was greater destruction of capillary vessels within the
rabbits can produce nonbloody diarrhea (39, 536), suggesting connective tissue of the colonic mucosa.
other potential mechanisms of diarrhea besides binding of The significance of Stx in intestinal disease can differ accord-
toxin to villus tip cells. ing to the animal model used. In piglets, the presence or
Support for the role of Stx in intestinal disease also comes absence of Stx made no difference to the diarrhea (664)
from studies with genetically mutated strains of other patho- whereas the extent and distribution of the A/E lesion was more
gens. Sjogren et al. (598) used a natural pathogen of rabbits, E. important in predicting intestinal symptoms (663). In an infant
coli RDEC-1, which normally causes nonbloody diarrhea and rabbit model, infection with an O157:H7 strain lacking Stx
A/E lesions. This strain, which contains the LEE pathogenicity showed the same changes in ion absorption and secretion as
island, does not normally produce Stx and appears to be a did infection with an O157:H7 expressing Stx (392). In this
lapine version of a human EPEC strain. These investigators model, development of the A/E lesion and infiltration of the
added a bacteriophage expressing Stx1 to RDEC-1 and orally intestinal tissues with PMNs was crucial to the development of
inoculated young rabbits with the hybrid strain. The infection diarrhea (187, 392). The overall conclusion to be drawn from
with the Stx-positive strain (RDEC-H19A) was much more these different studies and different models is that the ability of
severe than that with the toxin-negative strain, with more se- EHEC to produce the A/E lesions is probably sufficient to
cause nonbloody diarrhea but that Stx is essential for the de- the relative contributions of direct cytotoxic action by Stx and
velopment of bloody diarrhea and hemorrhagic colitis. indirect action via cytokines to the pathology seen in the kid-
(iii) Stx in HUS. Stx produced in the intestine is assumed to ney and other organs remains to be established.
translocate to the bloodstream, although toxin has never been EAST1. EAST1, first described in EAEC (see below), is also
detected in the blood of HUS patients. In polarized intestinal found in many EHEC strains. In one study, all 75 O157:H7
epithelial cells in vitro, Stx moves across the epithelial cell EHEC strains possessed the astA gene encoding EAST1, usu-
monolayer without obvious cellular disruption, probably ally with two gene copies in the chromosome (564); other
through a transcellular, rather than paracellular, pathway (5). Stx-producing strains, including 8 (89%) of 9 O26:H11 strains
Damage of the intestinal epithelium by Stx, bacterial lipopoly- and 12 (52%) of 23 non-O157/O26 strains also carried the astA
saccharide (LPS), or other inflammatory mediators could also gene (564). The significance of EAST1 in the pathogenesis of
aid translocation of the toxin to the bloodstream. This possi- disease due to EHEC is unknown but it could possibly account
bility is supported by the fact that patients with bloody diarrhea for some of the nonbloody diarrhea frequently seen in persons
due to E. coli O157:H7 are more likely to develop HUS than infected with these strains.
are those with nonbloody diarrhea (261). Although there is no Enterohemolysin. The 60-MDa plasmid commonly found in
animal model that reproduces the renal histopathology char- O157:H7 strains contains genes encoding a hemolysin (termed
acteristic of HUS following intestinal administration of toxin, enterohemolysin) (572). Enterohemolysin is found in nearly all
intravenous administration of Stx1 or Stx2 in a rabbit model O157:H7 strains and is widely distributed among non-O157

produces vascular lesions in the intestine and central nervous Stx-producing E. coli strains. In Germany, approximately 90%
system, organs where there are high concentrations of the Gb3 of all Stx-positive strains isolated from patients possessed
receptor (39, 536). (Rabbit kidneys lack Gb3 [85], and little or genes encoding this hemolysin (56). In one study of O111:H-
no renal damage is seen in this model [536].) Gb3 is present in Stx-producing E. coli strains isolated from patients, the entero-
high concentrations in human renal tissue (85), and Stx is hemolytic phenotype was observed in 16 (88%) of 18 O111:H-
cytotoxic to human renal endothelial cells in vitro (401). The strains isolated from patients with HUS but in only 4 (22.2%)
typical human renal histopathology includes swollen glomeru- of 18 patients with diarrhea without HUS (573). Patients with
lar endothelial cells and deposition of platelets and fibrin HUS develop antibodies to enterohemolysin (572), but there
within the glomeruli (452). Stx is believed to damage the glo- are no data indicating that it is involved in pathogenesis of
merular endothelial cells, leading to narrowing of capillary disease. Enterohemolysin belongs to the RTX toxin family,
lumina and occlusion of the glomerular microvasculature with members of which are expressed by uropathogenic E. coli,
platelets and fibrin (401). The decreased glomerular filtration Pasteurella haemolytica, and other human and animal patho-
rate is presumably responsible for the acute renal failure that gens (42). The gene encoding the hemolysin (ehxA) has ca.
is typical of HUS. Traversal of the occluded microvasculature 60% identity to the hlyA gene encoding hemolysin expressed by
could also injure erythrocytes to produce the fragmented cells uropathogenic E. coli (42, 573). The role of enterohemolysin is
that are characteristic of HUS. still subject to speculation. Lysis of erythrocytes in vivo would
Epidemiological data suggest that Stx2 is more important release heme and hemoglobin, which enhance the growth of E.
than Stx1 in the development of HUS (reviewed in reference coli O157:H7 and could serve as a source of iron (see below).
261). It has been reported that O157:H7 strains that express In addition to lysing erythrocytes, the toxin lyses bovine but not
Stx2 alone are more likely to be associated with progression to human leukocytes (42). Two other genetically distinct phage-
HUS than are strains producing Stx1 alone or, curiously, both encoded hemolysins, called Ehly1 and Ehly2, have been re-
Stx1 and Stx2 (515). However, other studies have not found a ported to be produced by many Stx-producing E. coli strains
statistically significant association of Stx2-only strains and pro- (58, 571, 624) but there are no data to suggest in vivo expres-
gression to HUS (126). There are also experimental data for sion or any role in pathogenesis for these hemolysins.
both cultured renal endothelial cells (401) and mice (682) Intestinal adherence factors. The only potential E. coli
suggesting that Stx2 is more potent in inducing cytotoxicity O157:H7 adherence factor that has been demonstrated to play
than is Stx1. As noted above, Stx2 is not a homogeneous class a role in intestinal colonization in vivo in an animal model is
of toxins, and even among variants with identical B subunits the 94- to 97-kDa OMP intimin, encoded by the eae gene. In
(i.e., Stx2 variants other than Stx2e), sequence differences conventional and gnotobiotic piglets, O157:H7 strains produce
within the A subunit can lead to differences in lethality for extensive A/E lesions in the large intestine, featuring intimate
mice (440, 507). Some Stx2 variants can be activated by intes- adherence of the bacteria to the epithelial cells. In contrast,
tinal mucus, and this activation may be associated with greater O157:H7 strains specifically mutated in the eae gene no longer
lethality (440). produced A/E lesions and, indeed, did not appear to colonize
Although the simplest mechanism for HUS involves direct any intestinal site (162, 434, 663). Additional support for a role
cytotoxic action of Stx on renal endothelial cells, there are also in human disease is seen with the anti-intimin immune re-
several studies that support a role for cytokines in this process. sponse seen in HUS patients (436) and, by extrapolation, the
Purified Stx has been reported to induce the expression of reduced virulence in volunteers of an EPEC strain mutated in
proinflammatory cytokines such as tumor necrosis factor alpha eae (see above). As noted above for the intimin of EPEC, the
(TNF-a) and IL-6 from murine peritoneal macrophages (646) sequence of the putative receptor binding portion (C-terminal
as well as specific synthesis of TNF in the kidney (278). TNF-a end) of intimin can vary among serotypes, and it was hypoth-
and IL-1b can enhance the cytotoxic effect of Stx on human esized that this sequence difference could account for the fact
vascular endothelial cells in vitro (345, 400), and these two that EPEC is a small bowel pathogen while EHEC is a large
cytokines, as well as TNF-b and bacterial LPS, have been bowel pathogen (711). In piglets, wild-type EPEC strains cause
shown to induce the expression of Gb3 and increase the bind- A/E lesions in both the small and large intestine while wild-
ing of Stx to human endothelial cells (672). In clinical studies, type EHEC strains cause A/E lesions only in the large intes-
elevated levels of IL-6 are found in the serum and urine of tine. When the cloned EPEC eae was introduced into the
HUS patients, and the levels of IL-6 correlate with the severity EHEC eae mutant, the hybrid EHEC strain expressing the
and outcome of disease (344, 673). These studies strongly EPEC intimin caused A/E lesions in both the small and large
indicate that cytokines are involved in the disease process, but intestine. In addition, the volume of diarrhea was greater in
piglets infected with the EHEC strain expressing the EPEC serotype O5:H-, loss of this plasmid had no effect on adhesion.
intimin than in those infected with wild-type EHEC, indicating Dytoc et al. (180) reported in vivo data supporting the involve-
that the degree of diarrhea increased with the amount of small ment of this plasmid in intestinal adherence after oral inocu-
bowel colonization. With the ability to change the site of in- lation of adult rabbits. In this study, E. coli K-12 strain HB101
testinal colonization by substituting EPEC and EHEC genes, containing this plasmid adhered to rabbit intestinal cells
these studies demonstrate that, at least in the piglet model, the whereas HB101 without the plasmid did not adhere. In both
intimin protein is essential for specific colonization in the large rabbit (392) and gnotobiotic piglet (664) models of disease, the
intestine. E. coli O157:H7 produces fimbriae which might aid presence or absence of this plasmid made no difference to the
intestinal adherence (24, 222, 331, 657), but no cloned fimbrial amount of diarrhea, the intestinal histopathology, or the intes-
genes have been reported. tinal ion transport. However, a serious limitation to establish-
The existence of intestinal adherence factors distinct from ing such a role is that there is no suitable animal model that
intimin is suggested by the isolation of Stx-producing E. coli reproduces all aspects of the disease, from intestinal inocula-
strains of serotypes other than O157:H7 that lack the eae gene tion to bloody diarrhea, to renal involvement (reviewed by
but are still associated with bloody diarrhea or HUS in hu- Gyles [272]). In these rabbit and piglet studies, the presence or
mans. In vitro adherence to cultured epithelial cells has been absence of Stx also made no difference, further highlighting the
shown for eae-negative strains of serotypes such as O113:H21 limitations of animal models. (However, a very promising
(181, 583, 692), but no specific candidate adhesins have been model of HUS involving greyhound dogs has recently been

identified. Other candidate adhesins have been reported for E. reported [283].) Epidemiological evidence suggests a stronger
coli O157:H7, but none have been well characterized or spe- correlation of the presence of this plasmid with the develop-
cifically demonstrated to play a role in adherence in vivo. ment of HUS rather than diarrhea. As described above, the
Sherman et al. (591) reported that a 94-kDa OMP distinct enterohemolytic phenotype encoded on this plasmid was ob-
from intimin (399) mediated adherence to HEp-2 epithelial served in 16 (88%) of 18 O111:H- strains isolated from patients
cells, but no further characterization of this factor has been with HUS but in only 4 (22.2%) of 18 O111:H- strains isolated
reported. The potential role of LPS in adhesion has been from patients with diarrhea without HUS (573).
examined, and two groups of investigators have reported that Despite the uncertainty about the significance of plasmid
the O157 polysaccharide side chain was not involved in adher- pO157 in disease, it is in fact widely distributed among human
ence to cultured epithelial cells (68, 130). In fact, loss of the O EHEC isolates. The initial study by Levine et al. (390) of the
side chain actually increased rather than decreased the adher- distribution of this plasmid among human isolates (mostly
ence to cultured epithelial cells (68, 130). Tarr (636) reported from North America) found that 99% of 107 O157:H7 strains
that a protein with homology to the iron-regulated gene A possessed the plasmid, as did 77% of 44 O26:H11 strains.
(irgA) of V. cholerae (248) was involved in adherence of E. coli pO157 was also found in 81% of 26 Stx-positive strains of
O157:H7 to HeLa cells, but no further details for this adhesin, serotypes other than O157:H7 and O26:H11 (390). A subse-
designated Iha for (IrgA-homologue adhesin), have yet been quent study with a different strain collection from Europe
reported. Type 1 fimbriae were suggested to be involved in the showed similar results, with the plasmid being present in 60%
adherence of some EHEC strains on the basis of inhibition by of Stx-positive strains of serotypes other than O157:H7 and
growth in mannose (176, 551, 695), but a subsequent study O26:H11 (692). Another study in Germany found pO157 in ca.
reported that growth of EHEC in mannose resulted in 90% of all Stx-producing E. coli isolates from patients (56). In
catabolite repression (476). Catabolite repression is a global contrast to the high frequency of the plasmid in human iso-
regulatory system that controls the expression of numerous lates, only a minority of Stx-positive strains of non-O157:H7
bacterial functions including carbon utilization, motility, sporu- serotypes isolated from cattle possess this plasmid (37).
lation, antibiotic biosynthesis, and, possibly, adherence of In addition to the 94- to 104-kb pO157 plasmid, a number of
EHEC, although mechanisms for the latter are unknown (476). other plasmids ranging in size from 2 to 87 kb have been found
pO157 plasmid. All strains of O157:H7 contain a highly in strains of E. coli O157:H7 (692). However, no correlation
conserved plasmid, designated pO157 (574), which varies in has been seen with possession of any of these plasmids and
size from 93.6 to 104 kb (575). This plasmid is also present in clinical disease.
O26:H11 strains and is present in most but not all Stx-produc- Iron transport. E. coli O157:H7 contains a specialized iron
ing E. coli strains isolated from humans (56, 390). A 3.4-kb transport system which allows this organism to use heme or
fragment of this plasmid, subsequently shown to encode en- hemoglobin as an iron source (377, 450, 655). A 69-kDa outer
terohemolysin (572), was developed by Levine et al. (390) as a membrane protein encoded by the chuA (E. coli heme utiliza-
diagnostic probe for EHEC. In addition to the enterohemoly- tion) gene is synthesized in response to iron limitation, and
sin and potential adherence factors described above, this plas- expression of this protein in a laboratory strain of E. coli was
mid encodes a catalase-peroxidase, whose function is unknown sufficient for utilization of heme or hemoglobin as the iron
(93). A possible role of this plasmid in the suppression of source (655). A gene homologous to chuA is also present in
production of an exopolysaccharide has also been suggested strains of Shigella dysenteriae I but not, interestingly, in other
(222). Shigella spp. or in other Shiga toxin-producing E. coli strains
The role of this plasmid in the pathogenesis of disease due such as those of serotype O26:H11 (450, 655). The growth of E.
to EHEC is unknown. In vivo and in vitro studies have re- coli O157:H7 is stimulated by the presence of heme and he-
ported conflicting results on the role of the plasmid in adher- moglobin (377, 450), and the lysis of erythrocytes by one or
ence to epithelial cells. Karch et al. (331) first reported that more of the hemolysins reported for this pathogen could re-
pO157 was required for the expression of fimbriae and adhe- lease these sources of iron, thereby aiding infection.
sion to epithelial cells. Other investigators have reported that Other potential virulence factors. O157 LPS (as well as LPS
loss of this plasmid either decreased adhesion (657), enhanced from other bacteria) enhances the cytotoxicity of Stx on human
adhesion (324), or had no effect on adhesion (222). A study by vascular endothelial cells in vitro, but its effects in vivo are not
Hall et al. (276) reported that for one EHEC strain of serotype clear. There is one report (487) that E. coli O157:H7 can
O103:H2, loss of this plasmid coincided with reduced adhesion invade cultured intestinal cell lines, but a later report (435)
to cultured epithelial cells while for another EHEC strain of disputed these findings, showing that O157:H7 strains were no
more invasive than E. coli strains from the normal flora. Fur- tralia, Chile, and South Africa, and non-O157:H7 EHEC se-
thermore, there is no in vivo evidence that invasion occurs in rotypes are often more important than O157:H7 serotypes.
humans or in animals. There is an interesting phenomenon observed in developing
countries wherein EHEC is much less frequently isolated than
Epidemiology other diarrheagenic E. coli strains, such as ETEC or EPEC.
The much lower incidence of EHEC in developing countries
The initial description of the EHEC epidemiology has been than in developed countries does not appear to be a reporting
provided in detail above and illustrates the complexity of rec- artifact, since EHEC strains have been actively sought in sev-
ognizing and controlling new and emerging pathogens. The eral studies (12, 141).
epidemiology of EHEC continues to unfold, and control of Animal reservoir. Stx-producing E. coli can be found in the
disease due to this organism has remained elusive. It is doubt- fecal flora of a wide variety of animals including cattle, sheep,
ful that we have seen the peak of this epidemic. The salient goats, pigs, cats, dogs, chickens, and gulls (57, 263, 315, 683a).
features of EHEC epidemiology include a reservoir in the However, the great majority of these strains are of serotypes
intestinal tract of cattle and other animals; transmission by a other than O157:H7 and are of questionable pathogenicity (see
wide variety of food items, with beef being a major vehicle of below). The most important animal species in terms of human
infection; and a very low infectious dose, enabling high rates of infection is cattle. High rates of colonization of stx-positive E.
attack and of person-to-person transmission. coli have been found in bovine herds in many countries (96,

Incidence. The large outbreaks involving hundreds of indi- 128, 263, 277, 686). These rates are as high as 60% but are
viduals are the infections that have garnered the most atten- more typically in the range of 10 to 25%. Stx-producing E. coli
tion, but sporadic EHEC infections comprise the major disease strains are usually isolated from healthy animals but may be
burden of this pathogen. The frequency of sporadic cases of associated with an initial episode of diarrhea in young animals
EHEC infection appears to be on the increase although in- followed by asymptomatic colonization. The isolation rates of
creased testing and reporting of EHEC complicates this con- O157:H7 are much lower than those of non-O157:H7 sero-
clusion. Sporadic infections due to E. coli O157:H7 appear to types. Surveys of U.S. dairy and beef cattle have found E. coli
be more common in Canada than in the United States (261), O157:H7 in 0 to 2.8% of animals, with the highest isolation
but such infections have already reached major proportions in rates reported from younger rather than older animals (195,
many areas of the United States. There appears to be a geo- 277, 686). The widespread distribution of STEC in animals
graphic distribution of EHEC infection, being more common corresponds to the occurrence of STEC in retail meats. In one
in northern than southern states of the United States and more study (168), E. coli O157:H7 was isolated from 3.7% of retail
common in western than eastern Canada. The CDC estimates beef, 1.5% of pork, 1.5% of poultry, and 2.0% of lamb samples.
the annual disease burden of E. coli O157:H7 in the United In another study on STEC in raw meats from grocery stores in
States to be more than 20,000 infections and as many as 250 Seattle, no O157:H7 isolates were recovered but a high prev-
deaths (84), but the failure of many clinical laboratories to alence of non-O157:H7 Stx-producing E. coli strains was found
screen for this organism greatly complicates any estimates. In in beef (23%), pork (4%), lamb (48%), veal (63%), chicken
some areas, E. coli O157:H7 is more frequently isolated from (12%), turkey (7%), fish (10%), and shellfish (5%) (554). The
routine stool specimens than are Shigella spp. and it is the isolation of these organisms from fish and shellfish suggests
second or third most frequently isolated pathogen after that cross-contamination of food can occur within grocery
Campylobacter and/or Salmonella spp. (261, 501). Notably, on stores, and at least two outbreaks in Connecticut involving
a nationwide basis, E. coli O157:H7 is the pathogen most delicatessen products were attributed to deficiencies in super-
frequently isolated from stool specimens with visible blood market hygiene practices (33).
(599). Some studies suggest that O157:H7 may cause only 50 to The reservoir of EHEC in farm animals is further docu-
80% of all EHEC infections (see below), and because EHEC mented by seroepidemiologic surveys, which show that the
strains of serotypes other than O157:H7 are not routinely incidence of elevated O157 LPS antibody levels in serum is
sought, the overall incidence of EHEC infections is very diffi- threefold higher in Canadian dairy farm families than in urban
cult to estimate. families (12.5 and 4.7%, respectively) and that the incidence of
The CDC has recently initiated the Foodborne Disease Ac- elevated levels of anti-Stx1 antibodies is sixfold higher in farm
tive Surveillance Network (FoodNet) to continually assess the families (42 and 7.7%, respectively) (534). The continual ex-
burden of foodborne disease at several surveillance sites in the posure of farm families to these organisms has been linked to
United States (114). The results from 1996, the first year of subclinical infections that serve to immunize against disease
surveillance, show nationwide incidence rates for Campy- (694). The immunizing benefit of farm life was seen in an
lobacter spp. (25 per 100,000 population), Salmonella spp. (16 outbreak of disease due to Stx-positive E. coli O111:NM on a
per 100,000), Shigella spp. (9 per 100,000), E. coli O157:H7 (3 dairy farm in which only visiting urban relatives of residents
per 100,000), Yersinia spp. (1 per 100,000), Listeria spp. (0.5 per were ill while members of the resident farm family remained
100,000), and Vibrio spp. (0.2 per 100,000) (114). These results healthy (342).
correlate with previous estimates that E. coli O157:H7 is the Transmission. EHEC can be transmitted by food and water
fourth most costly foodborne disease in the United States and from person to person. Most cases are caused by ingestion
(414). This surveillance network has recently been expanded to of contaminated foods, particularly foods of bovine origin. In
include cases of HUS, which will provide additional informa- the United States, ingestion of undercooked hamburgers, pre-
tion on the burden of disease due to non-O157:H7 EHEC in pared in a restaurant or in the home, has been a particularly
the United States (405). important cause of outbreaks (reviewed in references 261,
In addition to its importance in North America, EHEC is an 263). The largest outbreak so far reported in North America
important pathogen in Europe and Japan. In these developed involved hamburgers from a fast-food restaurant chain in De-
countries of the northern hemisphere, there is a distinct sea- cember 1992 and January 1993. Of the 732 affected individuals
sonality to infection, with most sporadic cases being reported in Washington, Idaho, Nevada, and California, 195 were hos-
in the summer. EHEC is also an important pathogen in some pitalized and 4 died (45, 261). Contamination of the hamburg-
countries of the southern hemisphere such as Argentina, Aus- ers implicated in these outbreaks was the result in part of
modern food-processing technology. Beef from thousands of (46). One adult patient infected with an Stx-positive E. coli O
cattle raised on hundreds of farms is ground together in a rough:H21 strain was still culture positive 5 months after the
single hamburger plant, which then distributes frozen patties to termination of a 4-week course of diarrheal illness (493). A
thousands of restaurants in several states. study of long-term E. coli O157 shedding in HUS patients in
Consumption of pink ground beef is also an important risk Germany reported a median duration of 21 days with a range
factor for sporadic infections with E. coli O157:H7 in Canada of 5 to 124 days (336). In two patients in this study, an Stx gene
(380) and the United States (438). In one study, hamburgers was apparently lost over several weeks of fecal shedding, which
prepared at home were shown to be an important source of was accompanied by a change in the pulsed-field gel electro-
sporadic O157:H7 infections (438), but the authors of this phoresis (PFGE) pattern of the O157 isolates. The change in
study suggest that many infections resulted not from direct PFGE patterns due to loss of stx genes has implications for the
ingestion of undercooked hamburgers but from cross-contam- use of molecular epidemiological techniques for distinguishing
ination of other food items by food preparers who did not wash this organism.
their hands after handling raw ground beef. Other foods of Non-O157:H7 serotypes. Most outbreaks of EHEC infection
bovine origin, including roast beef and raw milk, and other have been caused by O157:H7 strains, suggesting that this
types of meats, including meat from porcine, avian, and sheep serotype is in some way more virulent or more transmissible
sources, have also been directly linked to outbreaks (261, 263). than other serotypes. Nevertheless, other serotypes of Stx-
The spectrum of vehicles implicated in disease due to EHEC producing E. coli have been implicated in both sporadic dis-

is expanding far beyond the initial hamburger-associated out- ease and outbreaks, and the incidence of disease due to other
breaks. Recent outbreaks have been linked to consumption of serotypes is considered to be on the rise (315). Estimation of
mayonnaise (261), unpasteurized apple juice (53, 426), and the true incidence of disease due to non-O157:H7 EHEC is
fermented hard salami (112). The last two vehicles illustrate a greatly complicated by the need to detect these infections by
notable ability of E. coli O157:H7 to grow in foods of low pH the presence of Stx or stx genes, since these serotypes are
under conditions where other pathogens would not survive. usually sorbitol positive and there are no convenient media
This organism can adapt to acidic conditions to allow it to such as sorbitol MacConkey agar (see below) that will reliably
survive at pH 3.4 for several days (48, 391, 716). Raw vegeta- screen for them. In addition, distinguishing the true pathogens
bles such as lettuce have been the incriminated vehicle in within this group is further complicated by the fact that mere
several outbreaks (454), and in the recent outbreak in Japan expression of Stx is apparently not sufficient to confer virulence
involving over 9,000 cases, uncooked radish sprouts were im- but that other virulence factors, some known and some un-
plicated in the majority of cases (628). In most of these cases, known, are also necessary. Two recent reviews have focused
the fruits or vegetables were believed to be contaminated with specifically on the non-O157:H7 serotypes of EHEC (315,
cattle feces and the low infectious dose necessary for disease 638).
(see below) combined with the consumption of uncooked E. coli strains belonging to over 200 serotypes can express
foods permitted infection. Two recent outbreaks of O157:H7 Stx, but within most serotypes, both Stx-positive and Stx-neg-
infection in Michigan and Virginia were associated with con- ative strains can be found (315). More than 50 of these sero-
sumption of alfalfa sprouts (113). The seeds used to grow the types have been associated with bloody diarrhea or HUS in
sprouts were from the same distributor, and the infections humans. The most common non-O157:H7 serotypes associated
were probably caused by contaminated alfalfa seeds rather with human disease include O26:H11, O103:H2, O111:NM,
than contamination during the sprouting process. Water and O113:H21 (263). At least 10 outbreaks due to these or-
sources, including recreational water (347), well water, and ganisms (including one due to Stx-producing C. freundii) have
even a municipal water system (627), have also been associated been reported in Japan, Germany, Italy, Australia, the Czech
with outbreaks. The outbreak due to the municipal water sys- Republic, and the United States (reviewed in reference 315).
tem, which affected 243 individuals and caused four deaths, These outbreaks have involved 5 to 234 individuals, and for
resulted from an improperly repaired water system which al- most of them the source of infection could not be determined.
lowed unchlorinated water to be widely distributed. The con- It has been suggested that 20 to 25% of HUS cases in North
tamination of preserved foods, vegetables intended to be eaten America are due to non-O157:H7 EHEC (315). In many coun-
raw, and water is of particular concern since protection of the tries such as Chile (488), Argentina (396), and Australia (250),
public from these sources will be extremely difficult. non-O157:H7 EHEC serotypes account for the majority of
A very low infectious dose for EHEC infection has been HUS cases. Non-O157:H7 EHEC strains are also frequently
estimated from outbreak investigations (261). This number, on isolated from patients with nonbloody diarrhea. In a Belgian
the order of 100 to 200 organisms for infection, is similar to the study (520), 62% of the Stx-producing E. coli strains isolated
number required for Shigella infection and is consistent with from stool were non-O157:H7, compared to 32% that were
the numerous reports of person-to-person transmission in out- O157:H7. In Seattle, non-O157:H7 Stx-producing E. coli
breaks (45, 548) and in the institutional setting (46). A low strains were found in 1.1% of routine stool specimens, an
infectious dose is also consistent with waterborne transmission, isolation rate higher than that of Shigella or Yersinia spp. (0.2%
which has been documented in several reports. In one out- each) but lower than that of Campylobacter (2.5%) and Sal-
break of HC and HUS, E. coli O157:H7 was isolated from 21 monella (3.4%) spp. or E. coli O157:H7 (2.9%) (77). In Boston
individuals who swam in the same freshwater lake in Oregon (3) and Virginia (500), approximately half of all Stx-producing
(347). The source of the fecal contamination was hypothesized, E. coli isolates from patients were of non-O157:H7 serotypes.
but not proven, to be a toddler who was not yet toilet trained. However, in two case-control studies, non-O157:H7 Stx-pro-
The duration of shedding of E. coli O157:H7 in feces of ducing E. coli strains were recovered at similar rates from
infected patients varies widely. In a study of HUS patients in patients and healthy controls (91, 505). The recent introduc-
Seattle (639), 66% of patients initially positive for O157:H7 tion of commercially available kits to detect Stx should greatly
were negative 7 days after diarrhea began, even in the absence facilitate additional studies that are needed to assess the true
of antibiotic therapy. In a study of children with O157:H7 incidence of non-O157:H7 EHEC serotypes in human disease.
infections in Minnesota child day care facilities, the median The significance of isolating a non-O157:H7 Stx-producing
duration of shedding was 17 days with a range of 2 to 62 days E. coli strain from human stool is not as certain as that of
isolating an O157:H7 strain. There are several reports of pa- ical features of disease due to EHEC and refer readers to these
tients from whom such a strain was isolated who had high reviews for citations in the primary literature.
levels of antibody to the O157 LPS in serum (reviewed in Clinical disease. The incubation period of EHEC diarrhea is
reference 638). The presence of these antibodies suggests that usually 3 to 4 days, although incubation times as long as 5 to 8
the patient was coinfected with a non-O157:H7 strain and an days or as short as 1 to 2 days have been described in some
O157:H7 strain which was not isolated from the stool culture outbreaks. The initial complaint is usually nonbloody diarrhea,
and which may have caused the disease symptoms. In contrast although this is preceded by crampy abdominal pain and a
to the probable significance of isolating a non-O157:H7 strain short-lived fever in many patients. Vomiting occurs in about
from human specimens, the isolation of non-O157:H7 Stx- half of the patients during the period of nonbloody diarrhea
producing E. coli from foods is of doubtful significance. As and/or at other times in the illness. Within 1 or 2 days, the
noted above, some surveys have isolated non-O157:H7 Stx- diarrhea becomes bloody and the patient experiences in-
producing E. coli from up to 63% of retail meat samples, which creased abdominal pain. This stage usually lasts between 4 and
would cause an enormous disease burden if all such strains 10 days. In severe cases, fecal specimens are described as “all
were pathogenic. blood and no stool” (539). In most patients, the bloody diar-
There are at least two additional virulence factors that can rhea will resolve without apparent sequelae, but in about 10%
help distinguish pathogenic from nonpathogenic EHEC of patients younger than 10 years (and in many elderly pa-
strains: the A/E phenotype and the pO157 plasmid expressing tients), the illness will progress to HUS.

EHEC hemolysin. These characteristics can be detected with HUS is defined by a triad of hemolytic anemia, thrombocy-
the eae and pCVD419 probes, respectively, as described below. topenia, and renal failure; initial clinical manifestations in-
Several studies have examined the distribution of the eae and clude oligouria or anuria, edema, pallor, and, sometimes, sei-
pCVD419 sequences in human and animal strains (37, 57, 315, zures. Most patients will recover with appropriate supportive
398, 692). Although the results varied according to the strain therapy, but 3 to 5% of affected children will die and about 12
collection analyzed, most non-O157:H7 EHEC strains isolated to 30% will have severe sequelae including renal impairment,
from human disease are eae positive and/or pCVD419 positive, hypertension, or central nervous system manifestations (261,
compared to only a minority of such strains isolated from 515). There are no definitive markers for severity of disease,
animal and meat samples. In one study of 208 non-O157:H7 although host factors and therapeutic interventions are impor-
Stx-producing E. coli strains isolated from seven different spe- tant. Progression to HUS is more likely to occur in patients
cies of animals, only 1.4% of such strains were positive for eae infected with E. coli O157:H7 who experience bloody diarrhea,
(57). However, many non-O157:H7 isolates that are clearly fever, and elevated leukocyte count and who are very young or
associated with outbreaks or sporadic human disease do not old or were treated with antimotility agents (126, 416, 510).
possess either eae or the pO157 plasmid (77, 692) indicating The only bacterial factor that has been associated with more
that there must be additional, as yet unknown, virulence fac- severe disease is the expression of Stx2, as discussed above. It
tors that distinguish pathogenic from nonpathogenic non- should be noted that thrombotic thrombocytopenic purpura is
O157:H7 EHEC strains. also associated with O157 infection and appears to be related
pathogenetically to HUS (515).
Recurrent cases of HUS are quite uncommon. In a study of
Clinical Considerations HUS cases over a period of 20 years in Utah, HUS occurred
twice in the same patient in only 2.6% of all cases (595).
Few bacterial pathogens can routinely cause such striking Interestingly, although prodromal diarrhea is normal in typical
clinical syndromes in different organ systems as can EHEC in HUS, diarrhea is very uncommon among the small subset of
causing HC and HUS. The dual importance of this pathogen is recurrent HUS cases (595). This finding would suggest that the
reflected by the response of the medical community to the 1983 bacterial factors responsible for diarrhea invoke a protective
observation by Karmali (343) that linked the production of immune response against subsequent diarrheal disease due to
Stx/Vero cytotoxin to bloody diarrhea and HUS. This paper EHEC but that a primary EHEC infection may not evoke
has been referred to by a gastroenterologist as “one of the sufficient protective antitoxic immunity against a subsequent
most significant contributions to enteric microbiology of this EHEC infection (535).
century” (638) and by nephrologists as “arguably the most Isolation of EHEC from extraintestinal sites is very unusual,
important paper ever published on HUS” (330). but rare isolates of E. coli O157:H7 have been recovered from
The most extensive clinical observations have been made urine, blood, and the glans penis (261). These unusual isolates
with EHEC of the O157:H7 serotype. Some EHEC strains of were from patients with diarrhea. However, Tarr et al. (637)
non-O157:H7 serotypes cause clinical disease indistinguishable have recently reported an Stx-producing E. coli O103:H2 strain
from that caused by O157:H7, but as a group they appear to from a urinary tract infection that was unassociated with diar-
cause less bloody diarrhea and less HUS. Although nonbloody rhea. This infection developed into HUS and suggests that the
diarrhea, HC, and HUS are the most common clinical syndo- human uroepithelium can also permit the absorption of Stx.
mes, complications that can arise include cholecystitis, colonic Treatment. Treatment of EHEC disease is limited largely to
perforation, intussusception, pancreatitis, posthemolytic biliary supportive care. Although EHEC strains are generally suscep-
lithiasis, postinfection colonic stricture, rectal prolapse, appen- tible to a variety of antibiotics, there are no prospective studies
dicitis, hepatitis, hemorrhagic cystitis, pulmonary edema, myo- showing conclusively that the use of antibiotics alters the out-
cardial dysfunction, and neurological abnormalities (261, 635). come of disease. In a prospective study, Proulx et al. (526)
The frequencies of the various syndromes for diagnosed cases demonstrated a trend toward a lower incidence of HUS in
of O157:H7 infections include ca. 10% nonbloody diarrhea, ca. those receiving antibiotics. Consistent with this study, a retro-
90% HC, ca. 10% (of patients younger than 10 years) HUS, spective study conducted during the 1996 outbreak in Japan
and ,5% associated intestinal and extraintestinal complica- indicated that early treatment with one specific antibiotic, fos-
tions (635). Clinical aspects of disease due to EHEC have been fomycin, was associated with a reduced risk of HUS (631).
recently and extensively reviewed by multiple authors (84, 261, There are, however, retrospective studies which suggest that
474, 515, 625, 635). We will highlight the most important clin- patients who received antibiotics may be at greater risk of
developing HUS (108, 510); since these were not prospective, Prompt laboratory diagnosis and rapid epidemiological inves-
randomized trials, it could be that the patients who were most tigation and interventions prevented an estimated 800 addi-
severely ill were more likely to receive antibiotics. The use of tional primary cases (45). Diagnosis of EHEC infections also
antibiotics may be harmful for two potential reasons: first, lysis allows institution of appropriate isolation procedures for in-
of bacteria by some antibiotics leads to increased release of fected patients in the hospital or day care settings.
toxin, at least in vitro; second, antibiotic therapy could kill For the individual patient, the benefit of a prompt and ac-
other intracolonic bacteria, thereby increasing the systemic curate EHEC diagnosis can also be substantial. A variety of
absorption of toxin. While there is controversy about the use of incorrect diagnoses have been made for patients infected with
antibiotics, the use of antimotility agents such as loperamide is E. coli O157 including appendicitis, intussusception, primary
definitely not indicated in the management of disease due to inflammatory bowel disease, and ischemic colitis (261). Lack of
EHEC; there is evidence that the use of such agents can in- an accurate diagnosis has led to numerous unnecessary and
crease the risk for development of HUS, possibly by delaying expensive procedures including exploratory surgery, hemico-
intestinal clearance of the organism and thereby increasing lectomies, colonoscopies, barium enemas, and appendectomies
toxin absorption (126). (83). Tarr (635) has written:
Treatment of renal disease due to EHEC is primarily sup-
“The cost of materials needed to detect E. coli
portive, except for some experimental therapies currently be-
O157:H7 is less than $1.00 per stool sample, and the
ing evaluated in clinical trials. Current treatment regimens may

labor involved in review of the plates is minimal. In
include dialysis, hemofiltration, transfusion of packed erythro-
this era of managed care, if even one laparotomy or
cytes, platelet infusions, and other interventions as clinically
colonoscopy is averted by the timely diagnosis of E.
indicated. Severe disease may require renal transplant. A
coli O157:H7 infection or if even one severe second-
promising therapy now being evaluated in clinical trials is Syn-
ary case of E. coli O157:H7 infection can be pre-
sorb-Pk, which consists of a chemically synthesized analog of
vented, the cost of screening hundreds of stools for
Gb3, the receptor for Stx, coupled to diatomaceous earth (22).
this pathogen can be justified. Attempts at economy
This compound would be ingested by patients with bloody
are better directed at determination of appropriate
diarrhea in the hope that it could absorb toxin from the intes-
criteria for requesting a stool culture rather than at
tine and prevent the development of HUS. Initial phase I trials
limitation of the microbiological evaluation of speci-
have been promising (22), and phase III trials to assess efficacy
mens that are submitted.”
are in progress.
Vaccines. There are no currently available vaccines to pre- The CDC recommends that clinical microbiology laborato-
vent disease due to EHEC, but a number of experimental ries routinely culture stool specimens from persons with bloody
approaches are being investigated in animals. Vaccine devel- diarrhea or HUS for E. coli O157:H7 with SMAC agar (111).
opment has been severely hampered by the lack of an appro- (Other than the presence of blood, there are no obvious indi-
priate animal model wherein animals challenged orally with cations from a stool specimen whether to seek EHEC. Detec-
EHEC will develop HUS. A crucial antigen in any potential tion of fecal leukocytes, for example, is not helpful since fecal
vaccine is the Stx. Parenteral Stx toxoid vaccines have shown leukocytes are seen in fewer than half of all patients [635].)
protective effects in rabbits (64) and pigs (80). Attenuated V. The Association of State and Territorial Public Health Labo-
cholerae (4, 98) and Salmonella typhimurium (668) vaccine ratory Directors also recommends that clinical laboratories
strains that express StxB have been constructed. The V. chol- screen at least all bloody stool specimens for E. coli O157:H7
erae constructs have been administered orally to rabbits and with SMAC agar (83, 136). This group also recommends that
have engendered neutralizing serum antibodies and partial E. coli O157:H7 be a nationally reportable disease. (As of
protection from the enterotoxic effects of Stx (4). The intesti- February 1997, 42 states require O157 infections to be re-
nal adherence factor intimin has also been expressed in atten- ported [405].) A Consensus Conference panel of gastroenter-
uated V. cholerae strains (98a, 295). A parenteral vaccine spe- ologists, microbiologists, epidemiologists, food scientists, and
cific for O157 EHEC has been developed based on O157 infectious-disease specialists organized by the American Gas-
polysaccharide conjugated to protein carriers (365). An ideal troenterological Association Foundation concurred with this
broad-spectrum EHEC vaccine should probably engender recommendation and also recommended that because isola-
both systemic immunity against Stx and local intestinal immu- tion rates of E. coli O157:H7 from nonbloody stools approach
nity against intimin and other intestinal colonization factors. those of Salmonella, Shigella, and Campylobacter spp., all non-
bloody stools submitted for the examination of bacterial en-
Diagnosis and Detection teric pathogens also be cultured for E. coli O157:H7 (90). This
latter recommendation does not state that all nonbloody stools
General considerations. (i) Why and when to culture. With that enter a clinical microbiology laboratory should be cultured
increased emphasis on cost containment in the clinical micro- for E. coli O157:H7 but, rather, that once a decision has been
biology laboratory, there is economic pressure not to routinely made to culture potential pathogens from nonbloody diarrheal
test stool samples for EHEC. The current lack of a proven stools, E. coli O157:H7 should be sought together with other
treatment that is specific for EHEC infections might also di- enteric pathogens. Many considerations are involved in the
minish enthusiasm for adding yet another diagnostic test to the decision on how extensively the clinical microbiology labora-
laboratory. However, there are compelling clinical, public tory should analyze nonbloody stools to diagnose a diarrheal
health, and economic reasons for routinely screening for agent. A discussion of these considerations is beyond the scope
EHEC on at least some clinical specimens. of this review, but they have been recently reviewed (290).
The major public health reason for screening for EHEC is to Two years after the Association of State and Territorial
detect outbreaks. Once an outbreak is detected, appropriate Public Health Laboratory Directors recommended that clinical
public health interventions can be instituted to limit the num- laboratories screen at least all bloody stool specimens for E.
ber of cases and deaths. In the 1993 hamburger-associated coli O157:H7 with SMAC agar, Boyce et al. (83) surveyed 129
outbreak in the northwestern United States, 501 cases and laboratories in the United States that performed stool cultures
three deaths were reported in Washington State alone (45). and found that only 74 (54%) of these laboratories screened all
stools or all bloody stools for culture for E. coli O157:H7. tion of Stx-producing organisms or fecal Stx, and (iii) detection
Laboratories located in southern states were least likely to of elevated antibody levels to O157 LPS or other EHEC anti-
culture for O157:H7. Since reported infections with E. coli gens in serum. Detailed protocols for many of these methods
O157:H7 are more common in the northern states than in the have been reviewed by Smith and Scotland (603). Additional
southern states, these authors suggest that laboratory practices recent reviews for the diagnosis of EHEC from clinical and
may contribute to apparent regional differences in the inci- food specimens have been published by Strockbine et al. (623)
dence of O157:H7. Some laboratories that screened all stool and Meng and Doyle (444), respectively. In this section, we will
samples found no or only rare isolates of O157:H7 among present an overview of diagnostic methods and will review
thousands of stool cultures and therefore discontinued the methods for subtyping strains and determining the presence of
practice (290, 610). Boyce et al. (83) recommend that all lab- other virulence factors such as eae or the pO157 plasmid.
oratories at least perform a pilot study during the warmer Although these latter methods are not typically used in clinical
months in which all stools submitted for culture are screened microbiology laboratories, they can provide additional impor-
for E. coli O157:H7. Depending on the background incidence tant information for diagnostic or epidemiological investiga-
of this organism, laboratories can then adjust their policies to tions.
screen all stools all the time, only bloody stools, or some The isolation of E. coli O157:H7 or other Stx-producing E.
compromise formula to account for seasonal differences in coli strains from stool specimens depends upon culturing early
incidence. As an example of such analysis, one group in Wash- in the course of disease. Unfortunately, many patients are not

ington State (where the incidence of infection is high) calcu- extensively evaluated until they have HUS symptoms, which
lated the testing cost per positive isolate as $183 if all stool usually begin several days after the onset of diarrhea. In one
samples are cultured, $173 if all stool samples with occult prospective study of HUS patients in Seattle, Tarr et al. (639)
blood are cultured, and $40 if only stools with gross blood are found that persons with HUS sought medical attention an
cultured (95). This group concluded that for small laboratories, average of 6.5 6 2.8 days after the onset of diarrhea. When
culturing only stools with gross blood provides reasonable sen- stool samples obtained within 2 days of the onset of diarrhea
sitivity and cost. The results of a large multicenter study sug- were cultured for E. coli O157:H7, the recovery rate was 100%.
gested that when the presence of fecal blood was used as the When stools were cultured 3 to 6 days or more than 6 days
sole criterion for culturing for O157 strains, only 3% of stools after diarrhea began, the recovery rate decreased to 91.7 and
would need to be cultured to detect 63% of all O157 infections 33.3%, respectively. Therefore, by the time HUS is manifest,
(599). If either the presence of visible blood or a history of two-thirds of the patients no longer have E. coli O157:H7 in
fecal blood were used, more than 90% of cases would be their stools (635). Conversely, as noted above, some patients
detected by culturing 22% of specimens. can shed the organism in their stool for weeks or months after
The decision whether to culture for E. coli O157:H7 with infection, thereby potentially requiring multiple cultures over
inexpensive SMAC agar is only the first decision to be made long periods for a single patient.
concerning the diagnosis of EHEC. A second decision must Tests for Stx-producing organisms (other than O157) or
also be made whether to screen for EHEC of serotypes other fecal Stx are generally more expensive than culture methods
than O157:H7. As discussed above, such serotypes are increas- for detecting O157 strains. In addition, this category of tests
ingly being recognized as pathogens. If the local incidence of may suffer from an additional complication due to the poten-
HUS is high and culture of bloody stools does not yield O157: tial instability of the phage carrying the stx genes. Karch et al.
H7, screening for EHEC of serotypes other than O157:H7 (335) tested the stability of stx genes in 45 strains that were stx
should be instituted. The decision to screen for non-O157:H7 positive upon initial isolation. After passage on Trypticase soy
EHEC involves the detection of Stx by methods that are agar or other common laboratory media, 15 of the 45 strains
considerably more expensive than the use of SMAC agar. had lost either stx1 or stx2 genes. The frequency of loss varied
These toxin-based methods and other diagnostic tests are according to strain, with strains of serotypes O2:H5 and O73:
reviewed below. H34 being particularly prone to toxin gene loss and strains of
(ii) Biosafety issues. E. coli O157:H7 and other Stx-positive O26:H11 being very stable. Loss of stx genes has also been
E. coli strains are perhaps the most dangerous enteric patho- observed in serial cultures from individual patients over the
gens that clinical microbiologists in developed countries are course of long-term shedding (336).
likely to encounter. There have been at least three cases of There are no common biochemical characteristics that are
laboratory-acquired infection with E. coli O157:H7 with seri- associated with the great majority of EHEC serotypes. How-
ous consequences including renal failure and hemorrhagic co- ever, there are some biochemical characteristics of E. coli
litis (79, 97, 531). No laboratory accident or obvious breach in O157:H7 that have been exploited in the isolation and identi-
technique was evident in any of these cases. This level of fication of this serotype. An important characteristic is that
biohazard is due to the extremely low dose required for infec- O157:H7 strains do not ferment D-sorbitol rapidly, in contrast
tion (see above) and the potent effects of Stx, which are not to about 75 to 94% of other E. coli strains (197, 198, 412). E.
limited to the intestine. The low infectious dose and the ex- coli O157 strains also do not ferment rhamnose on agar plates,
pression of Stx are reminiscent of those for S. dysenteriae I, whereas 60% of non-sorbitol-fermenting E. coli belonging to
which most microbiologists in developed countries are unlikely other serogroups ferment rhamnose (603). (O157 EHEC
to encounter. There also exists the potential for careless work- strains do ferment rhamnose within 1 day in standard tube
ers to transport the organisms home, where young children sugar fermentation tests, as opposed to agar plate tests [603].)
could be infected. In the United Kingdom, EHEC will be One report indicates that more than 90% of E. coli O157:H7
reclassified from hazard group 2 into hazard group 3, which strains give one of two unique biochemical profile numbers on
requires that the same laboratory containment and conditions a MicroScan conventional gram-negative identification panel
used to handle Salmonella typhi and Shigella dysenteriae type 1 that were not detected with other D-sorbitol-negative E. coli
be used to handle EHEC (525). strains (1). Another characteristic of E. coli O157:H7 that
(iii) Diagnostic methods. There are three major categories distinguishes it from most other serotypes of E. coli is the
of methods used to diagnose EHEC infections. These are (i) inability to produce b-glucuronidase, which hydrolyzes 4-meth-
isolation of E. coli O157 strains from stool samples, (ii) detec- yl-umbelliferyl-D-glucuronide (MUG) and related substrates
(649). E. coli O157:H7 also does not grow well at 44 to 45.5°C, after overnight incubation (when both a-hemolysin and en-
which is a temperature commonly used to grow E. coli from terohemolysins are evident).
food and water samples (529). Whatever plating and enrichment method is used, suspected
Culture techniques. The agar medium most commonly used EHEC colonies should be confirmed as E. coli by conventional
for the isolation of E. coli O157:H7 is SMAC agar (198, 412), tests and screened at least for the O157 LPS antigen as de-
which is available from multiple commercial sources. This me- scribed below. Escherichia hermanii is biochemically and sero-
dium contains 1% sorbitol in place of lactose in the standard logically similar to E. coli O157 (603) but can be distinguished
MacConkey medium. Sorbitol-nonfermenting colonies, indic- by cellobiose fermentation (E. coli is negative, and E. hermanii
ative of E. coli O157:H7, are colorless on this medium. Mul- is positive) and growth in the presence of potassium cyanide
tiple sorbitol-nonfermenting colonies (at least 3 and up to 10 (E. coli does not grow, and E. hermanii does grow). Stx-pro-
[260, 603]) should be selected for testing as potential E. coli ducing strains of E. hermanii have not been reported (603).
O157. SMAC agar is not generally useful for Stx-producing E. Presumptive identification of E. coli O157:H7 can be reported
coli strains of serotypes other than O157:H7 because there is for confirmed E. coli strains that are sorbitol negative on
no known genetic linkage between Stx production and sorbitol SMAC agar and agglutinate in O157 antiserum (261). Strains
fermentation. However, one study found that all 19 Stx-pro- can be forwarded to a reference laboratory for toxin testing
ducing E. coli strains isolated from HUS patients in Chile, and H typing.
including some O26, O111, and O55 strains, were sorbitol Immunoassays. Most of the immunological assays currently

negative (488). Failure to ferment sorbitol is also not tightly available for EHEC detect either O and H antigens or Stx. The
linked to other H types of the O157 serogroup, and several available immunoassays for the O and H antigens are limited
sorbitol-fermenting, Stx-producing O157:H- strains have been largely to detecting EHEC expressing O157 LPS and the H7
isolated from patients with HUS in Germany (270). The re- flagellar antigen, whereas toxin immunoassays are suitable for
covery rate of E. coli O157:H7 on SMAC agar can be improved detecting both O157 and non-O157 serogroups of EHEC.
by prior enrichment in selective broth for 4 h to overnight (i) O and H antigens. Colonies can be screened for the O157
rather than direct plating of stool specimens. GN Broth Hajna LPS directly from SMAC agar or after subculture, whereas
and trypticase soy broth supplemented with cefixime (50 ng/ screening for the H antigen may require passage through mo-
ml) and vancomycin (40 mg/ml) have both been used with tility medium before testing (see below). Antisera to the O157
success (332, 555). LPS and the H7 antigen have been incorporated into a variety
There are various modifications of SMAC agar that improve of diagnostic kits involving ELISA, latex reagents, colloidal
selectivity and differentiation. The inclusion of cefixime and gold-labeled antibody, and other formats and are available
tellurite (CT-SMAC agar) permits the growth of Stx-producing from numerous commercial sources (LMD Laboratories, Inc.,
E. coli O157:H7 and Shigella sonnei strains but partially or Carlsbad, Calif.; Oxoid Diagnostic Reagents, Basingstoke, En-
completely inhibits the growth of most of the other E. coli gland; Pro-Lab Diagnostics, Round Rock, Tex.; Remel Micro-
strains (713). Inclusion of novobiocin has also been reported to biology Products, Lenexa, Kans.; Difco Laboratories, Detroit,
increase the selection for E. coli O157:H7 (489). E. coli Mich.; Meridian Diagnostics, Cincinnati, Ohio; Kirkegaard &
O157:H7 also does not ferment rhamnose on agar plates, in Perry Laboratories, Inc., Gaithersburg, Md.; Neogen Corp.,
contrast to the majority of other sorbitol-nonfermenting Lansing, Mich.; Denka Seiken Co., Ltd., Tokyo, Japan). More
strains, and so the inclusion of both sorbitol and rhamnose in specialized reagents in which antibodies to O157 LPS are al-
SMAC agar plus cefixime (CR-SMAC) increases the propor- ready conjugated to fluorescein, peroxidase, or phosphatase
tion of colorless colonies on this medium that are actually of are also available (Kirkegaard & Perry Laboratories, Inc.). The
the O157:H7 serotype (119). (However, one recent study CDC has evaluated three commercial latex reagent kits for
found that some Stx-positive O157:NM strains did not grow on detecting the O157 antigen (Oxoid, Richmond Hill, and Re-
CT-SMAC [332].) The inability of E. coli O157:H7 to produce mel) and one latex reagent kit for detecting the H7 antigen
b-glucuronidase can be tested by using MUG (649). Hydrolysis (Remel) (611). The results of this study showed 100% corre-
of MUG by most non-O157:H7 E. coli strains produces a lation of the commercial O157 kits with the CDC reference
fluorescent compound, and this substrate has been incorpo- antisera. Some false-positive reactions with both the commer-
rated into some agar media (629). Various agar media based cial and CDC sera were seen with a strain of C. freundii and
on one or more of these properties are commercially available strains of Salmonella O group N. (The O157 antigen was pre-
(Rainbow Agar O157 [Biolog Inc., Hayward, Calif.], Fluoro- viously shown to be identical to the Salmonella O301 antigen
cult E. coli O157 agar [Merck, Darmstadt, Germany]). One [593].) The H7 reagent had a sensitivity of 96% and a speci-
medium, Rainbow Agar, contains chromogenic substrates that ficity of 100% compared to the standard tube agglutination
are specific for both b-galactosidase and b-glucuronidase, method with CDC reference antisera. Many strains that gave
yielding a spectrum of colored colonies ranging from black to initial negative reactions with the H7 reagent became positive
gray to red to blue to violet that purportedly differentiates when passed through motility medium to enhance motility
among E. coli O157:H7, E. coli O26:H11, other Stx-producing (199, 611). (Use of a PCR technique to detect the H7 gene can
E. coli serotypes, and Stx-negative E. coli. There is little pub- eliminate the need to pass strains [203].) The CDC study con-
lished information on side-by-side comparisons of these vari- cluded that the commercial latex reagents are good alterna-
ous media. tives to standard serologic methods for identifying the E. coli
A nonselective but differential plating medium is enterohe- O157 and H7 antigens when the manufacturer directions are
molysin agar (Unipath), which detects the enterohemolysin closely followed. Additional controls with non-O157 antiserum
expressed by about 90% of Stx-producing E. coli isolates from and latex control reagents should be included to rule out non-
humans (56) (see above). Stool samples are inoculated into specific agglutination.
tryptic soy broth, and the overnight growth is streaked onto the Commercially available ELISA kits to detect E. coli O157
plates (60). Isolated enterohemolytic colonies are then tested antigen directly in stool samples offer testing times of less than
for Stx production (see below). A disadvantage of this ap- 1 hour. These kits are accurate and easy to use in clinical
proach is the need to read and record hemolytic colonies 3 h laboratory settings (178, 502). In a study of 605 stool samples,
after plating (when only a-hemolysin is evident) and again Park et al. (502) found one such kit (E. coli O157 antigen
detection ELISA; LMD Laboratories, Inc.) to be more sensi- body has been used in both ELISA (497) and dipstick immu-
tive than direct plating on SMAC agar. Compared to a method noassays (356) to detect E. coli O157:H7 in foods; the capture
involving colony sweeps and immunofluorescence microscopy, antibody is a polyclonal antibody against the O157 LPS anti-
this ELISA had a sensitivity of 91.2% and a specificity of gen, and the detection antibody is the 4E8C12 monoclonal
99.5%. These authors found this technique to be faster and antibody. A kit containing this monoclonal antibody (EHEC-
easier than a direct immunofluorescence microscopy technique Tek; Organon Teknika, Durham, N.C.) is available for detec-
that they had previously reported for detecting O157 antigen tion of O157:H7 in food and environmental samples; addi-
directly in stool specimens (501). At least one other ELISA kit tional modifications to this assay have been reported to
for detecting O157 antigen in stools is commercially available improve the specificity of the reaction (316). A polyclonal
(Premier E. coli O157; Meridian Diagnostics, Inc.). Positive antibody that recognizes products of the pO157 plasmid of E.
results from this ELISA should be considered presumptive and coli O157:H7 has also been reported as a potential diagnostic
should be confirmed by culture, Stx tests, or PCR (502). technique (656) but is not commercially available.
(ii) Shiga toxins. Numerous immunological assays using var- (iv) Immunomagnetic separation. Because of the small
ious formats have been developed for the detection of Stx and numbers of E. coli O157 present in many stool samples, im-
Stx-producing E. coli. A comprehensive review of these tech- munomagnetic separation (IMS) with commercially available
niques, which have been used largely for research purposes, is magnetic beads coated with antibody against E. coli O157 has
beyond the scope of this review, and so we limit our discussion been used (Dynabeads anti-E. coli O157; Dynal, Inc., Lake

to assays that are commercially available. These tests generally Success, N.Y.). IMS has also been used to isolate this organism
take less than 1 h to complete and have the most potential to from food and water samples (20, 702, 710) and bovine feces
be adopted by clinical laboratories for detection of non- (120). For processing of human feces, a short preenrichment
O157:H7 EHEC. step in GN Broth Hajna (332) or buffered peptone water
The first (and, as of this writing, the only) test to be ap- supplemented with vancomycin, cefixime, and cefsulodin (118)
proved by the Food and Drug Administration is the Premier is followed by incubation with anti-E. coli O157 Dynabeads and
EHEC test (Meridian Diagnostics, Inc.). This test uses mono- magnetic separation. The paramagnetic bead-bacterium com-
clonal antibodies directed against Stx1 and Stx2 to capture the plex is then resuspended and plated on SMAC, CT-SMAC, or
antigens and polyclonal anti-Stx antibodies and horseradish CR-SMAC agar plates. In the study by Karch et al. (332),
peroxidase for detection (3). It is capable of detecting Stx which used a 4-h preenrichment in GN Broth Hajna prior to
antigen in bacterial cultures, food samples, and fresh or frozen IMS, O157 strains were detected at 102 CFU/g of stool in the
stool samples. Several studies have reported that it is easy to presence of 107 coliforms in the background flora. For 20 HUS
perform, sensitive, and very suitable for use in routine screen- patients with antibody levels in serum indicative of O157 in-
ing for non-O157 EHEC (3, 17, 500, 677). In one study, the fection, the IMS technique allowed the isolation of O157
Premier EHEC test was positive in 13 of 95 stool samples strains from 18 samples compared to 13 samples detected by
compared to 6 of 95 samples that were positive with SMAC stx PCR, 12 samples detected by stx colony hybridization, and
agar, although it was not as sensitive as PCR, which detected 7 samples detected by direct culture on SMAC or CT-SMAC
an additional 8 samples (3). Another study (348) found that the agar. These authors concluded that IMS is the most sensitive of
Premier EHEC test had a sensitivity and specificity of 100 and all detection techniques, even more sensitive than PCR, which
99.7%, respectively, for E. coli O157:H7 compared to the sen- required approximately 105 CFU of O157 organisms per g of
sitivity and specificity of SMAC agar, which were 60 and 100%, stool to yield a positive result (compared to 102 for IMS).
respectively. False-positive reactions due to the presence of Several other studies have also concluded that IMS is a highly
(Stx-negative) Pseudomonas aeruginosa in stools were initially sensitive technique for detecting E. coli O157 (54, 118, 145).
reported with this system (59), but this problem has been The commercially available magnetic beads are limited to iso-
corrected. This test, like other toxin antigen detection kits, lating O157 E. coli, but the IMS technique has been adapted to
does not identify Stx2e, which is much more common in strains isolate O111 strains in an outbreak setting (506).
associated with pig edema disease than with human disease (v) Free fecal cytotoxic activity. Testing for Stx activity in
(17). This kit is available as a 96-well microtiter plate with stool samples is an extremely sensitive method to detect the
detachable microwells so that 1 to 94 samples (plus 2 controls) current or recent presence of Stx-producing organisms (338).
can be tested at a current list price of $864 per plate. Several studies have shown the presence of free fecal cytotoxin
The VTEC-RPLA test (Oxoid, Inc.) is a reverse passive in samples in which conventional culture techniques failed to
latex agglutination test for detection of Stx1 and Stx2. Extracts yield any Stx-producing organisms (104, 332, 340). In studies in
of single colonies contain enough antigen to yield positive which the two techniques have been compared, testing for fecal
reactions in this test, which is performed in 96-well microtiter toxin is more sensitive than PCR (104, 332), but this discrep-
plates (60). This test has been combined with the enterohemo- ancy could be due to the presence of inhibitors of PCR in the
lysin test, in which stool samples are plated on enterohemoly- stools. For detection of E. coli O157, IMS appears to be more
sin agar and single, enterohemolytic colonies are tested for sensitive than testing for free fecal toxin (332), but the O157
toxin in the VTEC-RPLA test (60). An ELISA system to IMS assay will not detect Stx-producing strains of serogroups
detect Stx1 and Stx2, which uses antibodies against Stx1 and other than O157.
Stx2 to capture the antigen, has recently been developed by Numerous studies using this technique have contributed
LMD Laboratories. Another test using a Gb3 receptor capture much to our understanding of the epidemiology of disease due
system is the VeroTest (MicroCarb, Gaithersburg, Md.). This to Stx-producing strains other than O157:H7. However, testing
system purports to detect as little as 10 pg of Stx, a detection stool samples (or bacterial cultures) for cytotoxic activity in-
level equivalent to that seen with cell culture assays. volves cell culture assays that are unavailable or impractical for
(iii) Other antigens. A monoclonal antibody (4E8C12) that most clinical microbiology laboratories. Detailed protocols for
recognizes a 5- to 6-kDa OMP expressed by O157:H7 and this technique have been published (337, 603), but in brief,
O26:H11 strains has been used in the identification of EHEC diluted stool samples are centrifuged and a bacterium-free
strains of these serotypes (496). The function of the protein filtrate of the supernatant is added to cell cultures. The use of
recognized by the antibody is unknown. The monoclonal anti- Vero cells is recommended over HeLa cells, since HeLa cells
are less sensitive for some variants of Stx2. The cells are ob- stool with equal sensitivity for stx1 and stx2; the sensitivity was
served for up to 3 days for cytopathic effects, and any cytotoxic increased another 2 log units when a radiolabeled sxt probe
activity is confirmed by neutralization with specific anti-Stx was used to hybridize the PCR products in a Southern blot
serum (337). The same cell culture methods are used to test procedure. Paton et al. (505) used a broth enrichment for their
bacterial cultures for Stx activity. This test has largely been PCR procedure but found that ca. 50% of stool samples from
supplanted by the use of immunoassays to detect toxin in healthy children gave positive PCR results. Caprioli et al. (104)
stools, but little published information about the relative sen- reported that a stx PCR technique gave positive results for ca.
sitivities of the cell culture test and the commercially available 50% of stool samples that were positive for free fecal cytotoxin.
immunoassays is available. One recent study (348) reported Karch et al. (332) found that their PCR procedure had a
that the Premier EHEC test was at least as sensitive as fecal sensitivity of ca. 104 CFU per 0.1 g of stool, which was not as
cytotoxin assays. sensitive as IMS for detecting O157 (see above). These studies
DNA probes and PCR. Most DNA probes and PCR tech- indicate that PCR offers great potential for detecting EHEC in
niques for EHEC are directed toward the detection of genes stool samples but that additional work remains to determine
encoding Stx. While the presence of Stx-producing strains of the optimal stool-processing procedure and primer pairs.
any serotype in a clinical specimen is assumed to be significant, PCR techniques have also been used to detect low levels of
the mere presence of such strains of non-O157 serogroups in stx-containing organisms in ground beef and other foods (233,
food or other nonclinical samples is of uncertain significance. 696), although as noted above, the mere presence of non-

Frequent loss of stx genes upon subculture can also occur, as O157:H7 stx-positive strains in food samples is of unknown
described above. For these reasons, probes and PCR tech- significance. As few as 0.5 to 1 CFU of stx-containing E. coli
niques for additional EHEC virulence factors can often proper g of ground beef can be detected after enrichment in broth,
vide crucial information. and a quantitative PCR technique has been developed to de-
(i) Detection of stx genes. DNA fragment probes have been tect stx1 genes in such samples (696). Another PCR assay
used in research studies for nearly 10 years to detect EHEC designed for detecting E. coli O157:H7 in food and environ-
(475, 693). The use of fragment probes requires two different mental samples is commercially available (BAX for Screen-
probes, one for stx1 and one for stx2. With synthetic oligonu- ing/E. coli O157:H7; Qualicon, Wilmington, Del.). Whether
cleotide probes, at least two probes are usually used for stx1 this test detects genes encoding Stx, intimin (eae), H7 antigen
and stx2 and additional probes specific for stx2c and stx2e are (fliC), O157 LPS, the pO157 plasmid, b-glucuronidase, or
required to detect strains containing these genes (92, 270, 288, some other factor is proprietary information.
334, 648). One oligonucleotide probe that hybridizes to stx1, (ii) Detection of eae genes. The same eae probes used to
stx2, and stx2c at low temperatures (45°C) but only to stx1 at a identify EPEC can also be used to identify EHEC if they are
higher temperature (53°C) has been reported (92). derived from the highly conserved 59 end of the gene. The 1-kb
The PCR technique has been extensively used to detect stx fragment probe described by Jerse et al. (314) has been shown
genes either in stx-only techniques or in multiplex PCR tech- to be highly sensitive and specific in many studies. Other dou-
niques incorporating primers for eae, ehx, uidA, or fliC (110, ble-stranded eae probes or PCR assays have been developed to
223, 232, 284, 572) (see below). Numerous primers and PCR detect sequences in the 59 end of eae (234, 330a, 398, 691). As
protocols have been designed to amplify these genes (89, 233, noted above, the 39 end of the eae gene can vary greatly among
305, 318, 333, 491, 492, 505, 523, 549, 661). Some early studies serotypes. Specific oligonucleotide probes (398, 691) and PCRs
used a single primer pair to detect both stx1 and stx2 (333, 505), (234, 330a, 398) have been developed to detect sequences in
but most methods now include two primer pairs that yield the 39 end of eae genes from EPEC O127:H6 (234, 578),
different-sized products for stx1 and stx2. Not all primer pairs EHEC O157:H7 (234, 398, 578, 691), and EHEC O111 strains
designed for stx2 will detect all variants of this gene (530), and (398). Interestingly, stx-negative O157:H45, O157:H8, and
specific primer pairs for stx2c (549, 661) and stx2e (216, 318, O157:H39 strains possess 39 eae sequences nearly identical to
647) have been designed. A combination of PCR and restric- those found in EPEC O127:H6 but quite different from the
tion fragment length polymorphism (RFLP) analysis has been O157:H7 eae sequences (578, 691). Primers for eae have been
used to distinguish stx2c from stx2 genes (104, 549, 661). In this combined with primers for stx1 and stx2 in multiplex PCRs
technique, PCR is performed with a single primer pair capable (223, 284).
of amplifying both genes and the amplified product is digested (iii) Detection of the pO157 plasmid/hemolysin gene. The
with a restriction endonuclease that will yield different frag- pO157 plasmid is present in nearly all O157:H7 strains and
ment sizes for stx2 and stx2c. many other EHEC strains (see above). A 3.4-kb fragment
The various PCR techniques are highly sensitive and specific probe empirically derived from this plasmid (called CVD419)
when used with bacterial colonies or cultures, but the use of was reported by Levine et al. (390). This probe has been
PCR for direct analysis of stool samples suffers from the same extensively used to identify and characterize EHEC strains
problems with background and inhibitory factors that are seen despite the unknown function of the sequences contained on
with other applications of PCR to stool samples. Several stud- this fragment. This fragment was subsequently shown to con-
ies have used PCR to detect stx genes in stools (89, 104, 145, tain the ehxA (hlyA) genes encoding the EHEC hemolysin
332, 505, 530). Using spiked stool samples, Ramotar et al. (574). Oligonucleotide probes and PCR techniques to detect
(530) could detect stx1-containing organisms at 102 CFU per genes encoding the hemolysin on the pO157 plasmid have also
0.1 g of stool and stx2-containing organisms at 107 CFU per been developed (223, 572).
0.1 g of stool; the reason for the greatly reduced sensitivity for (iv) Detection of other genes. PCR techniques to detect the
the stx2 genes was unknown. These investigators found that the gene (fliC) encoding the H7 antigen have been developed (203,
PCR technique was more sensitive than SMAC agar for the 232). The fliC primer pair has also been combined with primers
detection of E. coli O157:H7 but was less sensitive than were for stx and eae in a multiplex PCR to allow the specific iden-
cell culture assays for free fecal cytotoxin. This study also tification of E. coli O157:H7, O157:NM, and other EHEC
found excellent correlation between the PCR results and the strains (232). As noted above, O157:H7 strains do not produce
hybridization of 50 colonies per sample with stx probes. Brian b-glucuronidase. A multiplex PCR which incorporates primers
et al. (89) reported a sensitivity of ca. 105 CFU per 0.1 g of for the mutant glucuronidase gene (uidA) together with primer
pairs for stx1 and stx2 in a mismatch amplification mutation (38). Bender et al. (47) recently used PFGE to subtype E. coli
assay to identify O157:H7 and the specific stx genes has been O157:H7 strains in Minnesota and found that routine surveil-
developed (110). lance by this technique “can identify outbreaks that are not
Strain subtyping. E. coli O157:H7 strains form a highly detected by traditional methods and can ascertain whether
conserved clone that shows low genetic diversity in “house- sudden increases in reported cases are due to sporadic isolated
keeping” genes as assessed by multilocus enzyme electrophore- cases or to one or more outbreaks.” The CDC is establishing a
sis analysis (688). This clone is only distantly related to other national electronic database of PFGE subtypes to facilitate
serotypes of Stx-producing E. coli (688–690) and in fact is more recognition of outbreaks (36).
closely related to O55:H7 EPEC strains, which Whittam et al. Serodiagnosis. Detection of a serum immune response is not
(690) have proposed as the progenitor of the O157:H7 patho- usually used for the diagnosis of infections due to other diar-
genic clone. Because the O157:H7 clone is so highly conserved, rheagenic E. coli strains, but serodiagnostic techniques can
a variety of techniques have been used to differentiate strains provide valuable diagnostic information for EHEC infections,
of this serotype for epidemiological studies. These techniques particularly since many cases of HUS are not recognized until
can also be used to differentiate EHEC strains of serotypes after fecal shedding of the organism has ceased. Unfortunately,
other than O157:H7, but serotyping is a more useful marker there is no single antigen that is ideal for use in serodiagnostic
for these strains than it is for O157:H7 strains. As with other assays. Stx represents an obvious choice for an important an-
bacterial pathogens, the use of molecular epidemiological tigen produced in vivo. However, numerous investigations of

techniques can be crucial in investigations of outbreaks due to the serological response to Stx have yielded disappointing re-
EHEC, particularly in establishing whether cases are linked to sults. A curious phenomenon is that sera from most individuals
a common source or whether they represent sporadic and without any history of infection with Stx-producing E. coli
unrelated cases. strains contain a substance that is capable of neutralizing Stx2.
Plasmid profiles have been used in several studies to distin- This nonspecific neutralizing activity has been attributed to a
guish strains of O157:H7, and although they have provided high-density LPS present in serum rather than to specific anti-
useful information, the authors usually conclude that other Stx1 antibodies (105). When ELISAs rather than neutraliza-
methods provide better discrimination (444, 503). Sequence tion tests are conducted to detect antibodies to either Stx1 or
variation within stx genes has been used to distinguish different Stx2, only a minority of HUS patients showed a response to
strains by using specific stx oligonucleotide probes or PCR these toxins (35, 122, 342, 594). Similar results are seen in
techniques (104, 216, 288, 318, 549, 648, 661). Phage typing can patients infected with Stx-producing S. dysenteriae I (358a), and
separate O157:H7 strains into 66 different phage types (227, it has been suggested that this lack of response may be because
354), but this technique is available only in reference centers Stx is cytotoxic to human B lymphocytes in vitro (131). How-
that possess the typing phage. A combination of phage typing ever, Reymond et al. (534) report that the frequency of VT1
and specific stx gene probes has been used in the United (Stx1) antibodies was about sixfold higher in dairy farm resi-
Kingdom as a rapid and discriminatory typing system for in- dents than in urban residents. Since dairy farm residents have
vestigating E. coli O157 epidemiology (227). Ribotyping has sustained exposure to the bovine reservoir of STEC, these
also been applied to this pathogen, but this technique was results suggest that multiple exposures are required to induce
unable to differentiate among O157:H7 strains (417). Random detectable antibody responses to Stx, thus greatly limiting the
amplified polymorphic DNA PCR (RAPD-PCR, also called value of this antigen for serodiagnosis. Reymond et al. (535)
arbitrarily primed PCR) has been successfully used to discrim- have recently developed a Western blot technique to detect
inate among O157:H7 strains (284, 404). This technique uses serum antibodies to Stx. This technique offers several advan-
low-stringency PCR amplification with arbitrarily chosen oli- tages over ELISA and neutralizing-antibody assays, but the
gonucleotide primers and allows any laboratory with a PCR authors conclude that it is still not a suitable assay for diag-
machine to distinguish strains of this serotype. Screening for nosing a recent EHEC-associated illness.
antibiotic resistance patterns may also provide useful subtyping For serodiagnostic approaches, the most widely studied an-
data, but because the use of antibiotics is not indicated for tigen is the LPS (35, 69, 121, 125). Chart et al. (125) showed
treatment, such information is useful only for epidemiological that in one study of 60 patients with HUS, Stx or a Stx-pro-
purposes. ducing E. coli strain could be detected in only 23% of fecal
Since EHEC strains contain one or more large (60- to 70-kb) specimens whereas an IgM response to the O157 LPS was
l-like bacteriophages that contain the stx genes, variations in detected in 73% of these patients. In an epidemic situation, an
phage content and chromosomal insertion sites can lead to immunoassay based on IgG responses to LPS was over 90%
strain differences that can be detecting by Southern hybridiza- sensitive and specific for patients with recent culture-con-
tion or PFGE techniques. Hybridization of genomic digests of firmed infection (35). Antibodies to the other antigen of the
E. coli O157:H7 strains (separated by conventional gel elec- O157:H7 serotype, the H7 flagellum, were not detected in any
trophoresis) with labeled l DNA provides useful and discrim- of the HUS patients studied by Chart et al. (124). In the same
inatory RFLP patterns (264, 503, 553), but this technique study, some patients showed responses to OMPs, but this re-
yields very complex patterns that can complicate the analysis of sponse was found to be due to contaminating LPS comigrating
large numbers of strains (552). Hybridization of Southern blots with the OMPs. Although the O157 LPS is useful for serodi-
with labeled stx gene probes gives less complex but still very agnosis, it is difficult to prepare, and the response may be
sensitive RFLP patterns that are more readily interpreted nonspecific since O157 LPS shares epitopes with E. coli O44
(264, 552). and O55 LPS (402, 594) and the LPS of certain serogroups of
PFGE has been used by several groups to investigate the Salmonella spp., Yersinia enterocolitica, Brucella abortus, and V.
molecular epidemiology of O157:H7 infections (38, 87, 368, cholerae non-O1 strains (reviewed in reference 704). Chart and
445), and an automated pattern recognition system to analyze Rowe (123) reported that five of nine individuals vaccinated
the data has been proposed (107). PFGE is a more sensitive with a parenteral cholera vaccine developed antibodies that
but more labor- and equipment-intensive technique than reacted with O157 LPS; such an antibody response would be
phage typing (38, 368), and the combination of these tech- considered indicative of infection with E. coli O157 if the
niques can offer advantages over the use of either system alone recent vaccination history were not known. Detection of serum
antibodies reactive with O157 LPS is also useless for detecting whereas of 134 probe-negative strains from asymptomatic con-
infection due to Stx-producing E. coli strains of non-O157 trols, only 20 (15%) were AA positive (P , 0.0002). Proposing
serogroups. Ludwig et al. (402) tested sera from HUS patients a new category of diarrheagenic E. coli, the authors coined the
against a battery of purified LPS preparations including O157, term enteroadherent-aggregative E. coli (later shortened to
O26, O55, O111, and O128 and found that six of eight HUS enteroaggregative E. coli, and abbreviated EAggEC or simply
patients whose stool specimens yielded non-O157 STEC iso- EAEC) to describe strains expressing AA. Diffusely adherent
lates exhibited a serologic response to the homologous LPS. Of E. coli (DAEC) strains were not associated with diarrhea in
99 HUS patients with negative stool cultures for STEC, 82 had this study. Mathewson et al. (419) observed concurrently that
serological evidence of infection. E. coli strains that adhered to HEp-2 cells but were not of
A novel set of immunogenic proteins was recently reported EPEC serotypes were associated with diarrheal disease in adult
by Jarvis and Kaper (310). These proteins are the 24-kDa travelers to Mexico. Furthermore, these investigators demon-
EspA and the 37-kDa EspB proteins encoded in the LEE, as strated that one such strain was capable of causing diarrhea in
described above. Sera from HUS patients contained antibodies adult volunteers (418). In these reports, diarrheagenic E. coli
that reacted strongly with these proteins, but control patient strains that adhered to HEp-2 cells but were not of EPEC
sera did not react with these proteins. Antigens prepared from serotypes were termed “enteroadherent E. coli.” Vial et al.
O26 EHEC strains reacted as well as did antigens prepared unified these observations by demonstrating that the prototype
from O157:H7 strains. These proteins should be suitable for enteroadherent E. coli strain of Mathewson et al. exhibited the

serodiagnostic use in a wide variety of EHEC strains of many AA phenotype and was thus enteroaggregative (679). The term
different serotypes. Another protein encoded in the LEE, in- “enteroadherent” is still frequently used but should now be
timin, also engenders an antibody response in HUS patients replaced by the more precise terms enteroaggregative and dif-
(436), but the secreted EspA and EspB antigens engender fusely adherent (see below).
stronger antibody responses and are easier to prepare than EAEC strains are currently defined as E. coli strains that do
intimin. There are two potential limitations to the serodiag- not secrete enterotoxins LT or ST and that adhere to HEp-2
nostic use of these antigens. First, they can be used only in cells in an AA pattern. It is likely that this definition encom-
infections with strains containing the LEE, which appears to be passes both pathogenic and nonpathogenic clones, which share
the great majority of strains involved in human disease, and a factor(s) conferring a common phenotype. The heterogenous
second, there will be cross-reactivity with serum from patients pathogenicity of EAEC in humans has been confirmed in vol-
infected with EPEC. unteer studies and outbreak investigations (192, 245, 418,
470, 604).
ENTEROAGGREGATIVE E. COLI
Pathogenesis
The observation by Cravioto et al. (139) of HEp-2 adherence
by EPEC was seminal not only for the field of EPEC research The pathogenesis of EAEC infection is not well understood;
but also in that it served as the foundation for the discovery of however, a characteristic histopathologic lesion and several
at least two other categories of diarrheagenic E. coli. Scaletsky candidate virulence factors have been described.
et al. (568) and Nataro et al. (468) examined collections of E. Histopathology. Important clues to EAEC pathogenesis may
coli from studies of diarrhea in the developing world and be found by histopathologic examination of infected patients
found, like Cravioto et al., that most EPEC strains adhered to and animal models. EAEC strains characteristically enhance
HEp-2 cells. However, these investigators also showed that mucus secretion from the mucosa, with trapping of the bacteria
many E. coli strains that were not of EPEC serogroups adhered in a bacterium-mucus biofilm. Tzipori et al. (665, 666) fed a
to HEp-2 cells as well and, moreover, that the adherence series of EAEC strains to gnotobiotic piglets; although some of
phenotype was clearly distinguishable from that of EPEC. The these animals did not experience diarrhea, all animals tested
adherence pattern of EPEC was described as localized adher- developed an unusual mucoid gel closely adherent to the small
ence (LA), denoting the presence of clusters or microcolonies intestinal epithelium (Fig. 10). High-power examination of this
on the surface of the HEp-2 cells (568). In contrast, non-EPEC gel revealed the presence of large numbers of densely packed,
did not adhere in the characteristic microcolony morphology, aggregating bacteria. In addition, the intestinal epithelium dis-
instead displaying a phenotype initially described as diffuse played pitting of goblet cells, suggesting stimulation of mucus
adherence (DA); these E. coli strains were negative with the hypersecretion. Ligated rabbit ileal loops injected with EAEC
EAF probe (see the section on EPEC, above). also display pitting of goblet cells and embedding of aggregat-
With these observations in mind, Nataro et al. (466) exam- ing bacteria within a periodic acid-Schiff (PAS)-staining blan-
ined the HEp-2 adherence properties of E. coli isolated from ket (462, 679). Hicks et al. reported that EAEC strains adhere
the stools of 154 children with diarrhea and 66 healthy controls to sections of pediatric small bowel mucosa in an in vitro organ
in Santiago, Chile. In the course of this study, these investiga- culture model (286). In this series of experiments, as above,
tors were able to subdivide the “diffuse” adherence phenotype EAEC strains were observed to be embedded within a mucus-
into two further categories: aggregative and (true) diffuse (il- containing biofilm. The ability of EAEC to bind mucus has
lustrated in Fig. 1). Aggregative adherence (AA) was distin- been demonstrated in vitro (684), and volunteers fed EAEC
guished by prominent autoagglutination of the bacterial cells develop diarrhea which is predominantly mucoid (470). The
to each other; this often occurred on the surface of the cells, as role of excess mucus production in EAEC pathogenesis is
well as on the glass coverslip free from the HEp-2 cells. The unclear; however, the formation of a heavy biofilm may be
sine qua non of AA, however, was the characteristic layering of related to the diarrheagenicity of the organism and, perhaps, to
the bacteria, best described as a stacked-brick configuration. In its ability to cause persistent colonization and diarrhea.
diffuse adherence (DA), bacteria were seen dispersed over the In addition to the formation of the characteristic mucus
surface of the HEp-2 cell, with little aggregation and little biofilm, experimental evidence suggests that EAEC infection is
adherence to the glass coverslip free from the cells. Of 253 accompanied by cytotoxic effects on the intestinal mucosa. Vial
EAF probe-negative E. coli strains from Chilean diarrhea pa- et al. were the first to show that infection with EAEC strains in
tients, 84 (33%) exhibited the AA pattern of adherence, rabbit and rat ileal loop models (679) resulted in a destructive
lesion demonstrable on light microscopy. The lesion was char- OMP (683). However, genetic studies have been performed
acterized by shortening of the villi, hemorrhagic necrosis of the only with the AAFs. The relevance of AAFs to human disease
villous tips, and a mild inflammatory response with edema and is suggested by the fact that isogenic AAF/II-negative mutants
mononuclear infiltration of the submucosa. Transmission elec- of strain 042 are no longer able to adhere to human intestinal
tron microscopy showed normal microvillar architecture with- explants in vitro (146). Whether other adherence factors also
out invasion of enterocytes; both light and electron microscopy play a role in adherence to the mucosa or the development of
revealed adherent bacteria without formation of the A/E le- the EAEC biofilm remains to be demonstrated.
sion. EAST1. While studying the plasmid of strain 17-2, Savarino
Mucosal destruction has been demonstrated in autopsy spec- et al. identified an open reading frame encoding a 4,100-Da
imens of ileum from patients who died of EAEC persistent homolog of ST (561, 562). The product of this gene, EAST1, is
diarrhea during an outbreak in the malnutrition ward of Mex- a 38-amino-acid protein which features four cysteine residues,
ico City hospital (192). Recently, Hicks et al. have shown that unlike the six residues characteristic of E. coli ST. Of interest
EAEC cytotoxicity can be demonstrated in in vitro organ cul- is the observation that the eukaryotic membrane protein, gua-
ture by using pediatric intestinal biopsy specimens (286), and nylin, previously shown to have homology to ST, also contains
Nataro and Sears have shown that EAEC strain 042 elicits four cysteine residues. The role of EAST1 in secretion has not
cytotoxic effects on T84 cells (human intestinal carcinoma yet been determined, although EAST1 clones yield net in-
cells) in vitro (465) (Fig. 11). In the T84 cell model, EAEC- creases in short-circuit current in the rabbit mucosal Ussing

induced cytotoxicity was evidenced by a unique phenotype, chamber model (561). EAST1 has been found in ca. 40% of
characterized by vesiculation of the microvillar membrane fol- EAEC strains, yet toxin genes have not been found with higher
lowed by cell death and exfoliation of cells from the mono- frequency in patients with diarrhea than in healthy controls
layer. In addition, this effect was accompanied by increased (462). Interestingly, other E. coli categories, notably the EHEC
vacuole formation and separation of the nucleus from the strains, have been shown to elaborate EAST1 with higher
surrounding cytoplasm. The effects were seen most promi- frequency than EAEC strains do (see above), and the toxin was
nently in areas where EAEC organisms were adhering to the reportedly expressed by 38% of nonpathogenic E. coli strains
T84 cells. In both the T84 and in vitro organ culture systems, in one study (564).
the toxic effects require the presence of genes encoded on the Invasiveness. Benjamin et al. have suggested that some
65-MDa plasmid in addition to those encoding the adherence EAEC strains may invade intestinal epithelial cells in vitro
fimbriae. It should be noted, however, that not all EAEC (49). However, human intestinal explants infected with EAEC
strains elicit cytotoxic effects on intestinal mucosa. Such strain strains do not reveal internalization of the bacteria (286), and
heterogeneity may account for the inconsistent association of clinical evidence for a role for invasiveness is as yet lacking.
EAEC with diarrhea in epidemiologic investigations and Cytotoxins. The toxic effects observed in animal models,
volunteer studies. human intestinal explants, and T84 cells are not accompanied
Adherence. The AA phenotype of EAEC strains has been by internalization of the bacteria or by intimate attachment.
studied in great detail. Nataro et al. have identified a flexible, Therefore, several groups have attempted to identify secreted
bundle-forming fimbrial structure of 2 to 3 nm diameter, des- cytotoxins in EAEC. Eslava et al. have identified a 108-kDa
ignated aggregative adherence fimbriae I (AAF/I) (464). cytotoxin which elicits destructive lesions in the rat ileal loop
AAF/I mediates HEp-2 adherence and human erythrocyte (192). This protein was recognized by serum from patients
hemagglutination in strain 17-2. The genes for AAF/I are or- infected with EAEC in the Mexican outbreak described below.
ganized as two separate gene clusters on the 60-MDa plasmid Recent data suggest that this protein is a member of the au-
of strain 17-2, separated by 9 kb of intervening DNA (471, 472, totransporter family of proteins (322). This protein exhibits
563). Region 1 contains a cluster of genes required for fimbrial enterotoxic activity in the Ussing chamber (473). Moreover, it
synthesis and assembly, including the structural subunit of the may be the ca. 120-kDa EAEC supernatant protein shown by
fimbria itself. Nucleotide sequence analysis of the region 1 Baldwin et al. to elicit rises in intracellular calcium levels in
cluster suggests that AAF/I is a member of the Dr family of HEp-2 cells (29). A role for calcium as a second messenger of
adhesins, so called because they mediate adherence to the Dr an EAEC toxin is supported by the T84 cell model, in which
blood group antigen (563). Region 2 encodes a transcriptional microvilli are lost via membrane vesiculation (421, 465).
activator of AAF/I expression which shows homology to mem- Model of EAEC pathogenesis. Available data do not permit
bers of the AraC family of DNA binding proteins (472). The a full description of EAEC pathogenesis, yet several hypothe-
AAF/I fimbriae are bundle-forming fimbriae but do not show ses can be formulated. We propose a three-stage model based
homology to members of the so-called type 4 class of fimbriae on in vitro and animal data. Stage I involves initial adherence
(644). to the intestinal mucosa and/or the mucus layer. AAF/I and
By using immunogold electron microscopy and a DNA AAF/II are the leading candidates for factors that may facili-
probe derived from the biogenesis cluster of AAF/I, Nataro tate initial colonization. Stage II involves enhanced mucus
and coworkers have found that only a minority of EAEC production, apparently leading to deposition of a thick mucus-
strains express AAF/I (146). A second fimbria (designated containing biofilm encrusted with EAEC. The blanket may
AAF/II), which is distinct morphologically and genetically promote persistent colonization and perhaps nutrient malab-
from AAF/I, has now been identified (146). The genes encod- sorption. Stage III, suggested from histopathologic and molec-
ing AAF/II are also organized as two unlinked regions; how- ular evidence, includes the elaboration of an EAEC cytotoxin
ever, in this case, the fimbrial subunit is removed by more than which results in damage to intestinal cells. It is tempting to
15 kb from the required biogenesis gene cluster. This latter speculate that malnourished hosts may be particularly im-
cluster features the typical organization of the Dr family. Still paired in their ability to repair this damage, leading to the
more AA adhesins are likely to exist. persistent-diarrhea syndrome.
It has been suggested that AA may be due to factors other The site of EAEC infection in the human intestine has yet to
than the AAF (149, 683). Debroy et al. have suggested that an be clearly demonstrated. In vitro organ culture experiments
afimbrial outer membrane protein may be responsible for AA reported by Hicks et al. have shown that EAEC strains are able
in some strains (149); Wai et al. have implicated a 38-kDa to adhere to both small and large bowel mucosa (286). Tissue

FIG. 10. Interaction of EAEC with the intestinal epithelium. (A) Photomicrograph of the ileum of a gnotobiotic piglet fed EAEC strain 042. The arrow points to
the thick mucus gel adhering to the intestinal mucosa (666). (B) High magnification of the ileal mucosa of a piglet fed EAEC strain 17-2 as in panel A. Note the
aggregates of bacteria coating the villous surface. The villi are edematous, and the enterocytes themselves appear swollen. Reprinted from reference 665 with permission
of the publisher.
specimens in these studies were derived from pediatric patients sporadic diarrhea in Mexico, Chile, Bangladesh, and Iran (81,
and were not formalin fixed prior to incubation with EAEC 142, 282, 466).
strains. These features may explain the discrepant results ob- Gonzalez et al. studied the results of a prospective study of
tained by other investigators (364, 705). The short incubation 513 Venezuelan infants with diarrhea and 241 age-matched
period observed in some humans challenged with strain 042 controls (258). EAEC strains were found in 26.9% of diarrheal
(470) (less than 8 h) is also consistent with involvement of the patients and 15% of controls (P , 0.0004). The high attribut-
small bowel in diarrheagenicity. able risk of EAEC infection implicated this category as the
major E. coli pathogen in this cohort of infants.
Epidemiology Although most reports have implicated EAEC in sporadic
endemic diarrhea, a growing number of reports have described
A growing number of studies have supported the association EAEC outbreaks (192, 245, 604). Cobeljic et al. have described
of EAEC with diarrhea in developing populations, most prom- an outbreak affecting 19 infants in the nursery of a hospital in
inently in association with persistent diarrhea ($14 days). In Belgrade, Serbia, over a 9-day period in 1995 (129). Of the 19
several of these studies, EAEC cultured from the stool during infants, 12 yielded the same multiply antibiotic resistant EAEC
the first few days of diarrhea is predictive of a longer duration strain of serogroup O4, with an identical plasmid pattern, while
of illness (157, 282). 0 of 5 well neonates yielded this organism (P 5 0.02). In 16
The association of EAEC with diarrheal disease appears to babies the illness lasted 3 to 9 days (mean, 5.2 days), but in 3
be geographic. On the Indian subcontinent, five separate stud- infants, persistent diarrhea developed, lasting 18 to 20 days.
ies have been published which demonstrate the importance of Infants with diarrhea typically manifested liquid green stools;
EAEC in pediatric diarrhea (61–63, 326, 508). These studies in three, mucus was visibly apparent. There was no gross blood.
include hospitalized patients with persistent diarrhea (61), out- All but three infants required intravenous hydration, but they
patients visiting health clinics (63), and cases of sporadic diar- all survived. The source of infection was unclear.
rhea detected during household surveillance (62). Eslava et al. have described two outbreaks of EAEC persis-
Working in Fortaleza, Brazil, Guerrant and colleagues have tent diarrhea occurring in the malnutrition ward of a Mexico
demonstrated a consistent association between EAEC and the City hospital (192); infants who died in these outbreaks were
persistent diarrhea syndrome (196, 393, 521). In this area, found to have developed necrotic lesions of the ileal mucosa.
EAEC has been implicated in up to 68% of persistent diarrhea Smith et al. have reported four outbreaks of EAEC diarrhea in
cases (196), which represent a disproportionate share of diar- the United Kingdom in 1994 (604). These four outbreaks in-
rheal mortality. EAEC has also been implicated as a cause of volved 19, 10, 51, and 53 patients, respectively. Patients in

FIG. 11. Cytotoxicity of EAEC on T84 cells infected with EAEC strain 042. Note the aggregative adherence of bacteria to the apical membrane, associated with
a loss of microvilli and the rounding of the apical membrane. Reprinted from reference 465 with permission of the publisher.
these outbreaks experienced vomiting and diarrhea, usually leukocytes) and supranormal levels of IL-8 in the stool (619).
without fever. Persistent diarrhea occurred in a small number This observation suggests that EAEC infection may be accom-
of patients. Each of the outbreaks was associated with con- panied by a subtle form of mucosal inflammation.
sumption of a restaurant meal, but no single source could be
implicated. Smith et al. have also reported that at least three Detection and Diagnosis
outbreaks of diarrhea in the United Kingdom previously at-
tributed to EPEC were actually due to EAEC strains (604). In EAEC infection is diagnosed definitively by the isolation of
the United States, EAEC has been linked to diarrhea in human E. coli from the stools of patients and the demonstration of the
immunodeficiency virus-infected patients (425); however, the AA pattern in the HEp-2 assay. Analysis of small bowel aspi-
precise role for EAEC in AIDS diarrhea is unknown. rates has not increased the yield (196).
Perhaps even more significant than the association of EAEC HEp-2 assay. The HEp-2 assay remains the gold standard
with persistent diarrhea are the recent data from Brazil (619) for detection of EAEC. Although variations in the AA pattern
and Australia (188) that link EAEC with growth retardation in can be discerned, the presence of bacterial clusters in a
infants. In each of these studies, the isolation of EAEC from stacked-brick configuration should be used to identify EAEC
the stools of infants was associated with a low z-score for height strains.
and/or weight, irrespective of the presence of diarrheal symp- Different methods for performing the HEp-2 assay have
toms. Given the high prevalence of asymptomatic EAEC ex- been described (420, 466, 678) (see above). However, compar-
cretion in some areas (258, 466, 685), such an observation may ative studies (678) suggest that the technique as first described
imply that the contribution of EAEC to the human disease by Cravioto et al. (139) (i.e., a single 3-h incubation of bacteria
burden is significantly greater than is currently appreciated. with cells, without a change in medium during the course of the
assay) is best able to discriminate the three patterns (AA, DA,
and LA). It should also be stressed that AAF adhesins are
Clinical Features
maximally expressed in static Luria broth cultures at 37°C
The clinical features of EAEC diarrhea are becoming in- (464); therefore, the authors incubate all HEp-2 assay inocula
creasingly well defined in outbreaks, sporadic cases, and in the in this manner.
volunteer model. As described above, the outbreak in Serbia DNA probe. Several lines of evidence suggest that the large
suggested a watery, mucoid, secretory diarrheal illness with plasmids present in most EAEC strains have a high degree of
low-grade fever and little to no vomiting. Studies in India also DNA homology (41, 679). From strain 17-2, Baudry et al. (41)
suggest that the illness is most likely be manifested by watery, selected a 1.0-kb plasmid-derived Sau3a fragment that hybrid-
secretory diarrhea in the absence of fever and without gross ized with a fragment of similar size from the 65-MDa plasmid
blood (62, 508). However, grossly bloody stools have been of strain 042. In an evaluation of this fragment as a diagnostic
reported in up to one-third of patients with EAEC diarrhea probe, 56 (89%) of 63 EAEC strains (by HEp-2 assay) were
(142). In volunteers infected with EAEC, the diarrhea was positive with the EAEC probe by colony blot hybridization;
generally mucoid and of low volume without occult blood or only 2 of 376 strains representing the normal flora and other
fecal leukocytes; all patients remained afebrile. Steiner et al. diarrheagenic categories hybridized with the probe. Subse-
have found that a large percentage of patients excreting EAEC quent experience with the EAEC probe has revealed that the
have detectable fecal lactoferrin (a sensitive indicator of fecal correlation of probe positivity with AA varies by location. In
some studies, the correlation achieves the 89% sensitivity re- ation. The site of Shigella and EIEC infection is the colonic
ported by Baudry et al. (41, 326), while in other studies, the mucosa (558, 559). The interaction of EIEC organisms with
sensitivity may be substantially lower (196). The epidemiologic eukaryotic cells is shown in Fig. 12.
significance of probe-positive versus probe-negative strains is Genes necessary for invasiveness are carried on a 120-MDa
undetermined. The nucleotide sequence of the AA probe rep- plasmid in Shigella sonnei and a 140-MDa plasmid in other
resents a cryptic open reading frame which is adjacent to the Shigella serotypes and in EIEC (40, 560, 601). The invasion-
plasmid replicon (462). A PCR assay with primers derived related plasmid has been designated pInv. Figure 13 illustrates
from the AA probe sequence shows similar sensitivity and the present level of understanding of the plasmid-borne viru-
specificity (576). lence genes of EIEC and Shigella spp. Prominent among these
Other tests for EAEC. Several methods other than the genes are the mxi and spa loci, which encode a so-called type
HEp-2 and DNA probe assays have been described. Albert et III secretion apparatus (16, 19, 423, 676). This machinery is
al. (14) have reported that EAEC probe-positive organisms required for the secretion of multiple proteins which are nec-
display an unusual pellicle formation in Mueller-Hinton broth. essary for full pathogenicity. The Ipa proteins (IpaA to IpaD)
Similarly, when EAEC strains are grown in polystyrene culture are secreted proteins, of which IpaB, IpaC, and IpaD are
tubes or dishes at 37°C overnight without shaking, a bacterial effectors of the invasion phenotype (40, 274, 442, 443). IpaC
film is produced on the polystyrene surface and is easily visu- has been shown to promote the uptake of Shigella spp. into the
alized with Giemsa stain (462, 468). Both phenotypes are likely eukaryotic cell (415), whereas IpaB is thought to function in

to be due to high surface hydrophobicity, and both of these the lysis of the phagocytic vacuole (287) and in the induction of
techniques are convenient substitutes for the DNA probe in apoptosis in macrophages (719).
assaying EAEC. It should be emphasized, however, that until Shigella movement within the cytoplasm appears to be me-
epidemiologic studies show greater pathogenicity of probe- diated by the nucleation of cellular actin into a “tail” which
positive strains over probe-negative strains, the HEp-2 assay extends from one pole of the bacterium (7, 558, 675). As
should remain the gold standard for EAEC detection. additional actin is added to this structure, the bacterium is
The critical question in the management of patients from propelled through the cytoplasm, generally in the lateral direc-
whom EAEC strains are isolated is whether the isolate is tion. VirG (IcsA), is a surface protein which is essential for the
responsible for the symptoms. We accept that an EAEC strain nucleation of actin filaments and movement through the cyto-
is a likely cause of the patient’s diarrhea in three situations: (i) plasm and into adjacent cells (247, 558).
when the patient presents in the course of a documented Regulation of Shigella virulence is complex and features at
EAEC outbreak, (ii) when the patient’s isolate can be shown to least one regulatory cascade. VirR is a chromosomally en-
belong to one of the common EAEC serotypes associated with coded histone-like protein related to the drdX product (299,
disease (e.g., O44:H18); and/or (iii) when the patient exhibits 424). VirR apparently acts in concert with VirF, a transcrip-
persistent diarrhea and stools repeatedly yield an EAEC as the tional activator encoded on pInv (166). VirF exerts pleiotropic
predominant organism in the absence of another enteric effects, some of which function through the intermediate tran-
pathogen. scriptional activator VirB (652).
A PCR with oligonucleotide primers derived from the probe Enterotoxin production. As described below, both Shigella
sequence has also been developed (576). The sensitivity and and EIEC infections are characterized by a period of watery
specificity of the EAEC PCR are similar to those of the diarrhea that precedes the onset of scanty dysenteric stools
AA probe. containing blood and mucus. Indeed, in most patients with
EIEC infection and many with Shigella infection, only watery
ENTEROINVASIVE E. COLI diarrhea occurs. Nataro et al. (469) have cloned and sequenced
a plasmid-borne gene from EIEC (designated sen), which en-
EIEC strains were first shown to be capable of causing di- codes a novel protein with a predicted size of 63 kDa. A
arrhea in volunteer studies conducted by DuPont et al. in 1971 mutation in the sen gene causes a significant diminution of the
(175). EIEC strains are biochemically, genetically, and patho- enterotoxic activity of the parent strain. The purified Sen pro-
genetically related closely to Shigella spp.; like Shigella spp., tein elicits rises in Isc levels without having a significant effect
EIEC strains are generally lysine decarboxylase negative, non- on tissue conductance. A role for enterotoxins is unproven, but
motile, and lactose negative (88). their presence may explain the characteristic watery diarrhea
attributed to EIEC.
Pathogenesis
The precise pathogenetic scheme of EIEC has yet to be Epidemiology
elucidated; however, pathogenesis studies of EIEC suggest
that its pathogenetic features are virtually identical to those of Epidemiologic studies of EIEC mostly describe outbreaks.
Shigella spp. (for reviews of Shigella pathogenesis, the reader is In sporadic cases, many EIEC strains are probably misidenti-
referred to references 249 and 504). Both organisms have been fied as Shigella spp. or nonpathogenic E. coli strains. Docu-
shown to invade the colonic epithelium, a phenotype mediated mented EIEC outbreaks are usually foodborne or waterborne
by both plasmid and chromosomal loci. In addition, both EIEC (375, 413, 606, 659), although person-to-person transmission
and Shigella spp. elaborate one or more secretory enterotoxins does occur (279). The infective dose of EIEC in volunteers is
that may play roles in diarrheal pathogenesis. higher than that for Shigella spp. (176), and thus the potential
Invasiveness. The current model of Shigella and EIEC for person-to-person transmission is lessened. Endemic spo-
pathogenesis comprises (i) epithelial cell penetration, (ii) lysis radic disease occurs in some areas, generally where Shigella
of the endocytic vacuole, (iii) intracellular multiplication, (iv) spp. are also prevalent, but the epidemiologic features may be
directional movement through the cytoplasm, and (v) exten- different from those of Shigella spp. (353, 641). The incidence
sion into adjacent epithelial cells (249, 463, 558). When the of EIEC in developed countries is thought to be low, but
infection is severe, this sequence of events elicits a strong occasional foodborne outbreaks, such as one restaurant-asso-
inflammatory reaction which is manifested grossly as ulcer- ciated outbreak involving 370 people in Texas, do occur (259).

FIG. 12. Interaction of EIEC with epithelial cells. Like Shigella, EIEC strains invade intestinal epithelial cells, lyse the phagosomal vacuole, and move through the
cytoplasm, ultimately spreading to adjacent epithelial cells. This electron photomicrograph shows an EIEC organism free within the cytoplasm of an infected cell. Photo
courtesy of P. Small.
Clinical Considerations ment in plasmid pSF55) (600). Both of these probes are
virtually 100% sensitive and specific for EIEC strains that have
The clinical presentation of EIEC disease has been docu-
retained their virulence (253). A 21-base oligonucleotide de-
mented from outbreaks, endemic disease, and volunteer stud-
rived from ial is identical in sensitivity and specificity to the
ies (176, 375, 413, 606, 641, 659). EIEC infection presents most
polynucleotide probe (221). It should be noted, however, that
commonly as watery diarrhea, which can be indistinguishable
EIEC strains may lose all or part of the pInv plasmid on in
from the secretory diarrhea seen with ETEC. Only a minority
vitro passage or storage, and therefore strains should be hy-
of patients experience the dysentery syndrome, manifested as
bridized with the probe(s) as soon as possible after they are
blood, mucus, and leukocytes in the stool; tenesmus; and fever
shed in the feces.
(413, 606, 641). In two documented EIEC outbreaks, gross
A PCR assay with primers derived from ial was able to
blood was observed in 0 and 7% of persons infected (413, 606).
detect as few as 10 CFU of S. flexneri in stool without enrich-
Asymptomatic infections due to EIEC are probably unusual.
ment of the sample (219, 221); this compares with 1,000 CFU
detectable by DNA probe. The ial PCR is also effective in a
Detection and Diagnosis multiplex PCR system to identify EIEC strains simultaneously
EIEC strains can be difficult to distinguish from Shigella spp with other E. coli categories (219).
and from other E. coli strains, including nonpathogenic strains. Pal et al. have developed an ELISA to detect the ipaC gene,
In general, identification of EIEC entails demonstrating that which is contained on the Inv plasmid of EIEC and Shigella
the organism possesses the biochemical profile of E. coli, yet (499). Using this assay, these investigators identified EIEC and
with the genotypic or phenotypic characteristics of Shigella spp. Shigella strains isolated from the stools of children in Kuwait.
The classical phenotypic assay for Shigella and EIEC identifi- An advantage of this assay over other methods is that it does
cation is the Sereny (guinea pig keratoconjunctivitis) test, not require costly or highly sophisticated equipment and does
which correlates with the ability of the strain to invade epithe- not use live animals.
lial cells and spread from cell to cell (367). The ability to form
plaques in a HeLa cell monolayer also correlates with these DIFFUSELY ADHERENT E. COLI
virulence characteristics (441).
Two polynucleotide probes for the detection of EIEC and The term “diffusely adherent E. coli” was initially used to
Shigella spp. have been described. Probe pMR17 is a 17-kb refer to any HEp-2-adherent E. coli strain that did not form
EcoRI fragment derived from pInv of a Shigella flexneri sero- EPEC-like microcolonies. With the discovery of EAEC, most
type 5 strain (253, 701). ial is a 2.5-kb HindIII fragment iso- authors now recognize DAEC as an independent category of
lated from pInv of an EIEC strain (available as a cloned frag- potentially diarrheagenic E. coli.

FIG. 13. Genes involved in EIEC pathogenesis. Both plasmid and chromosomal genes are involved in conferring pathogenicity on EIEC strains; the genes depicted
in the figure have largely been elucidated in Shigella, but most, if not all, also exist in EIEC. The chromosomal locus kcpA activates transcription of the plasmid-borne
gene virG, which encodes an OMP required for directional movement through the cytoplasm. The plasmid-borne locus virK increases the surface expression of the VirG
protein through an unknown mechanism. A regulatory cascade of virulence genes has been described in which the plasmid locus virF interacts with the chromosomal
locus virR to regulate the transcription of the ipa gene cluster of secreted effector proteins. ipa regulation involves the intermediate regulator virB. The mxi and spa
loci encode a type III secretory system homologous to that of EPEC and EHEC.
Pathogenesis ation with diarrhea increased with age from 1 year to 4–5 years
Little is known about the pathogenetic features of DAEC- in Santiago, Chile (385). The reason for such an age-related
induced diarrhea. Bilge et al. have described the cloning and phenomenon is as yet unknown. Other epidemiologic features,
characterization of a surface fimbria in this strain, which me- such as the mode of acquisition of DAEC infection, are also as
diates the DA phenotype (65–67). The genes encoding the yet undetermined.
fimbria (designated F1845) can be found on either the bacte- Jallat et al. have shown that DAEC strains account for a
rial chromosome or a plasmid. The fimbrial genes show ho- large proportion of diarrheal cases among hospitalized pa-
mology to members of the Dr group of bacterial adhesins. tients in France who have no other identified enteropathogen
Benz et al. (51, 52) have described a 100-kDa OMP which is (307). This report suggests that DAEC strains may be impor-
associated with the DA phenotype in one strain of serotype tant diarrheal pathogens in the developed world.
O126:H27. The gene encoding this factor (designated AIDA-I)
has been completely sequenced. Use of a DNA probe specific Clinical Features
for AIDA-I suggests that this factor is expressed by a minority
of DAEC isolates (50). Few epidemiologic or clinical studies permit adequate
Yamamoto et al. (707), and Cookson and Nataro (133) have description of the clinical syndrome associated with DAEC
shown that DAEC strains are able to induce finger-like pro- infection. In one study, the majority of patients infected
jections extending from the surface of infected Caco-2 or with DAEC had watery diarrhea without blood or fecal
HEp-2 cells (Fig. 14). These projections apparently “embed” leukocytes (522).
the bacteria, providing protection against gentamicin but with-
out complete internalization. A role for this phenotype in Detection and Diagnosis
pathogenesis has yet to be demonstrated.
DAEC strains are defined by the presence of the DA pattern
in the HEp-2 adherence assay (574). A 700-bp polynucleotide
Epidemiology fragment derived from the daaC gene (66) has been used as a
Several recent studies have implicated DAEC strains as DAEC DNA probe; daaC encodes a molecular usher neces-
agents of diarrhea, while other studies have not recovered sary for expression of the F1845 fimbriae. Approximately 75%
DAEC strains more frequently from diarrheal patients than of DAEC strains from around the world are positive with this
from asymptomatic controls. An age-dependent susceptibility F1845 gene probe (462). However, due to the genetic related-
may explain this observation, because when populations are ness of F1845 to other members of the Dr family of adhesins,
stratified by age, the association of DAEC with diarrhea is false-positive reactions with the DA probe may occur, albeit
found only in children older than infants (34, 240, 266, 385). with unknown frequency. No PCR assay has yet been described
Levine et al. showed that the relative risk of DAEC in associ- to identify DAEC.
OTHER CATEGORIES OF E. COLI WHICH ARE

POTENTIALLY DIARRHEAGENIC
The six categories of E. coli that are described in the above

sections have each been implicated in several diarrhea studies
and are now generally accepted as diarrheagenic categories.
However, some studies have suggested that there may be still
other categories of diarrheagenic E. coli which are quite dis-
tinct from those described above. These will be briefly consid-
ered.
Gunzberg et al. (266) reported a study of Australian aborig-
ine children with diarrhea in which a significant association was
found between diarrheal illness and the presence of a cytotoxic
phenotype on HEp-2 cells in the HEp-2 assay. This phenotype,
typified by detachment of the cells from the glass within 3 h of
incubation with bacteria, was found in organisms regardless of
their adherence pattern. This pattern led the investigators to

propose that cell-detaching E. coli (CDEC) may be a new
category of diarrheagenic E. coli. Elliott et al. (188) have char-
acterized CDEC strains further and have shown that the de-
taching phenotype appears to be closely associated with the
production of the E. coli hemolysin and that these organisms
frequently secrete the cytotoxic necrotizing factor (CNF) but
do not belong to any of the recognized categories above. More-
over, these investigators have shown that CDEC strains are
able to elicit diarrhea and destructive histopathology in the
rabbit model. The role of CDEC in human diarrhea has yet to
be determined.
Two forms of CNF have been described, CNF1 and CNF2.
Both forms are large, monomeric proteins of 110 to 115 kDa
that induce multinucleation of eukaryotic cells (reviewed in
reference 495). The mechanism of action of CNF has recently
been described and appears to be a novel cytotoxic mechanism
(207). CNF induces a deamidation of glutamine 63 in the FIG. 14. Interaction of DAEC with epithelial cells. DAEC strain C1845
RhoA target protein, producing a glutamate residue at that incubated with HEp-2 cells for 8 h. Note the association of the bacteria with the
site. The deamidated RhoA is “locked on” and leads to an membrane and the formation of long finger-like projections emanating from the
cell. These projections wrap around the bacterium in a phenotype termed “em-
increase in the number of stress fibers within the target cell. bedding.” Invasion is rarely seen. Reprinted from reference 133 with permission
The affected cells become larger and syncytial and ultimately of the publisher.
die. How this mode of action might lead to diarrhea is not
known. Most CNF-producing E. coli strains isolated from di-
arrheal stools have been isolated from animals rather than
CONCLUSIONS
humans; most human isolates of CNF-producing E. coli have
been from extraintestinal infections (103, 151, 495). There is a Our perspective on intestinal E. coli has undergone a re-
clear need for case-control studies of CNF-producing E. coli to markable transformation in recent decades and undoubtedly
definitely establish whether these organisms are true human will continue to evolve. Once dismissed as a harmless inhabit-
pathogens. ant of the intestinal tract, E. coli is now seen as a pathogenic
Scott and Kaper (588) and Pickett et al. (516) have cloned species with remarkable versatility in its ability to cause disease
and characterized a gene from E. coli which encodes a cytole- in humans and animals. Outbreaks of disease due to E. coli can
thal distending toxin (CDT). It has been suggested that the affect thousands of individuals and can engender national and
mechanism of action of this toxin involves the GM2/M phase international headlines. Pathogen-specific virulence factors
growth arrest of the target cell, which leads to elongation and have been discovered that adversely affect a wide range of
ultimately to cell death (512). There is a growing CDT family eukaryotic cell processes including protein synthesis, cell divi-
as a result of reports that this toxin, or close homologs, is sion, ion secretion, and transcription. These factors are en-
produced by Campylobacter spp. (517), S. dysenteriae (490), and coded on a variety of mobile genetic elements such as plas-
Haemophilus ducreyi, an agent of genital ulcers (134). The mids, bacteriophages, transposons and pathogenicity islands;
mechanism of CDT action in diarrhea is not known, but par- this genomic plasticity implies ongoing reassortment of viru-
tially purified CDT from S. dysenteriae can produce profuse lence factors that complicates our efforts to categorize the
watery diarrhea in a mouse model of diarrhea (490). A con- various subgroups into sharply delineated pathotypes. This dy-
trolled study of CDT-producing E. coli infections in Bang- namism promises to present new challenges in the diagnosis,
ladeshi children found that although CDT-positive E. coli treatment, and prevention of E. coli infections.
strains were isolated from more children with diarrhea than
from healthy controls, this difference did not reach statistical
significance (13). Moreover, the CDT-positive strains isolated ACKNOWLEDGMENTS
from children with diarrhea usually also possessed virulence
properties of other diarrheagenic E. coli strains such as the This work was supported by Public Health Service grants AI-33096
ability to cause the A/E lesion. to J.P.N. and AI-21657 and AI-41325 to J.B.K.
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ERRATUM
Diarrheagenic Escherichia coli
JAMES P. NATARO AND JAMES B. KAPER
Center for Vaccine Development, Departments of Medicine, Pediatrics, and Microbiology & Immunology,
University of Maryland School of Medicine, Baltimore, Maryland 21201
Volume 11, no. 1, p. 148, Table 2: “SLTI” and “SLTII” should read “Stx1” and “Stx2,” respectively.
Page 165, column 2, line 20 from bottom: “Stx2” should read “Stx2e.”
Page 188: Reference 132 should read “Collington, G. K., I. W. Booth, and S. Knutton. Enteropathogenic Escherichia coli
(EPEC) infection rapidly modulates electrolyte transport in Caco-2 cell monolayers. Gut, in press.”
403

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CLINICAL MICROBIOLOGY REVIEWS, Jan. 1998, p. 142–201 Vol. 11, No.

Diarrheagenic Escherichia coli

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* Corresponding author. Mailing address: Center for Vaccine De-

ENTEROHEMORRHAGIC E. COLI .......................................................................................................................164

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INTRODUCTION methylene-blue agar, which selectively grow members of the

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ETEC STI TTAATAGCACCCGGTACAAGCAGG 492 GCTGTGAATTGTGTTGTAATCC 457

LT GGCGACAGATTATACCGTGC 581 GCGAGAGGAACACAAACCGG 581

EAF CAGGGTAAAAGAAAGATGATAA 214 TATGGGGACCATGTATTATCA 313

BFP AATGGTGCTTGCGCTTGCTGC 268 GCTACGGTGTTAATATCTCTGGCG 462

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EIEC ial CTGGATGGTATGGTGAGG 579 CCATCTATTAGAATACCTGTG 579

EAEC Plasmid CTGGCGAAAGACTGTATCAT 576 None

Pathogenesis (Fig. 4A) (622). The B subunits are arranged in a ring or

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epidemiologic studies suggest that CFA/I, CFA/II, or CFA/IV Epidemiology

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Pathogenesis Shiga toxins. The major virulence factor, and a defining

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OTHER CATEGORIES OF E. COLI WHICH ARE

The six categories of E. coli that are described in the above

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