INTRODUCTION

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Escherichia coli is the predominant facultative anaerobe of the human colonic flora. The organism typically colonizes the infant gastrointestinal tract within hours of life, and, thereafter, E. coli and the host derive mutual benefit (169). E. coli usually remains harmlessly confined to the intestinal lumen; however, in the debilitated or immunosuppressed host, or when gastrointestinal barriers are violated, even normal "nonpathogenic" strains of E. coli can cause infection. Moreover, even the most robust members of our species may be susceptible to infection by one of several highly adapted E. coli clones which together have evolved the ability to cause a broad spectrum of human diseases. Infections due to pathogenic E. coli may be limited to the mucosal surfaces or can disseminate throughout the body. Three general clinical syndromes result from infection with inherently pathogenic E. coli strains: (i) urinary tract infection, (ii) sepsis/meningitis, and (iii) enteric/diarrheal disease. This article will review the diarrheagenic E. coli strains, which include several emerging pathogens of worldwide public health importance, and will specifically focus on pathogens afflicting humans. We will particularly concentrate on the E. coli strains whose study has advanced most over the last decade, i.e., enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), and enteroaggregative E. coli (EAEC). Since the categories of diarrheagenic E. coli are differentiated on the basis of pathogenic features, emphasis will be placed on the mechanisms of disease and the development of diagnostic techniques based on virulence factors.

E. coli is the type species of the genus Escherichia, which contains mostly motile gram-negative bacilli within the family Enterobacteriaceae and the tribe Escherichia (55, 185).

E. coli can be recovered easily from clinical specimens on general or selective media at 37°C under aerobic conditions. E. coli in stool are most often recovered on MacConkey or eosin methylene-blue agar, which selectively grow members of the Enterobacteriaceae and permit differentiation of enteric organisms on the basis of morphology (32).

Enterobacteriaceae are usually identified via biochemical reactions. These tests can be performed in individual culture tubes or by using test "strips" which are commercially available. Either method produces satisfactory results.

For epidemiologic or clinical purposes, E. coli strains are often selected from agar plates after presumptive visual identification. However, this method should be used only with caution, because only about 90% of E. coli strains are lactose positive; some diarrheagenic E. coli strains, including many of the EIEC strains, are typically lactose negative. The indole test, positive in 99% of E. coli strains, is the single best test for differentiation from other members of the Enterobacteriaceae.

Serotyping of E. coli occupies a central place in the history of these pathogens (reviewed in reference 394. Prior to the identification of specific virulence factors in diarrheagenic E. coli strains, serotypic analysis was the predominant means by which pathogenic strains were differentiated. In 1933, Adam showed by serologic typing that strains of "dyspepsiekoli" could be implicated in outbreaks of pediatric diarrhea. In 1944, Kauffman proposed a scheme for the serologic classification of E. coli which is still used in modified form today.

According to the modified Kauffman scheme, E. coli are 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 currently. The presence of K antigens was determined originally by means of bacterial agglutination tests: an E. coli strain that was inagglutinable by O antiserum but became agglutinable when the culture was heated was considered to have a K antigen. The discovery that several different molecular structures, including fimbriae, conferred the K phenotype led experts to suggest restructuring the K antigen designation to include only acidic polysaccharides (394). Proteinaceous fimbrial antigens have therefore been removed from the K series and have been given F designations (494).

A specific combination of O and H antigens defines the "serotype" of an isolate. E. coli of specific serogroups can be associated reproducibly with certain clinical syndromes (Table 1), but it is not in general the serologic antigens themselves that confer virulence. Rather, the serotypes and serogroups serve as readily identifiable chromosomal markers that correlate with specific virulent clones (690).

One of the most useful phenotypic assays for the diagnosis of diarrheagenic E. coli is the HEp-2 adherence assay. The method has recently been reviewed in detail (160). This assay was first described in 1979 by Cravioto et al. (139) and remains the "gold standard" for the diagnosis of EAEC and diffusely adherent E. coli (DAEC). The HEp-2 assay has been modified often since its first description, including such variations as extending the incubation time to 6 h or changing the growth medium during the incubation. However, collaborative studies have shown that the assay performed essentially as first described provides the best ability to differentiate among all three adherent diarrheagenic categories (EPEC, EAEC, and DAEC) (678). The HEp-2 adherence assay entails inoculating the test strain onto a semiconfluent HEp-2 monolayer and incubating it for 3 h at 37°C under 5% CO₂. After this incubation time, the monolayer is washed, fixed, stained, and examined by oil immersion light microscopy. The three patterns of HEp-2 adherence (Fig. 1), localized adherence (LA), aggre gative adherence (AA), and diffuse adherence (DA), can be differentiated reliably by an experienced technician. However, the authors have found some strains which yield equivocal results reproducibly in the HEp-2 assay.

View larger version (87K): [in this window] [in a new window]   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.

Nucleic acid probes. The use of DNA probes for detection of heat-labile (LT) and heat-stable (ST) enterotoxins in ETEC revolutionized the study of these organisms, replacing cumbersome and costly animal models of toxin detection (455). Since then, gene probes have been introduced for all diarrheagenic categories. Two general methods are commonly used for nucleic acid probe specimen preparation. The first entails the inoculation of purified cultures onto agar plates to produce "colony" blots, in which 30 to 50 such cultures are inoculated per plate. After incubation, the bacterial growth is transferred to nitrocellulose or Whatman filter paper for hybridization (alternatively, the cultures can be grown directly on the nitrocellulose overlying an agar plate). The bacterial growth on the paper can be lysed, denatured, and hybridized with the probe in situ, and then a radiographic image is generated by exposure to X-ray film. Substantial experience by ourselves and others has demonstrated that the colony blot method is reliable and efficient. However, the use of this method requires that the E. coli strain first be isolated from the patient's stool, which introduces 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 that patients symptomatic with E. coli diarrhea generally present with the pathogenic strain as their predominant E. coli strain in the flora. Thus, studies in which three E. coli isolates are tested per diarrheal stool specimen will have acceptable sensitivity. If increased sensitivity is desired or if the study entails a large number of asymptomatic patients, isolating five isolates per specimen may be more appropriate.

PCR. PCR is a major advance in molecular diagnostics of pathogenic microorganisms, including E. coli. PCR primers have been developed successfully for several of the categories of diarrheagenic E. coli (listed in Table 2). Advantages of PCR include great sensitivity in in situ detection of target templates. However, substances within stools have been shown to interfere with the PCR, thus decreasing its sensitivity (615); several methods have been used successfully to remove such inhibitors, including Sepharose spin column chromatography and adsorption of nucleic acids onto glass resin (397, 615). Scrupulous attention to proper technique must be maintained to avoid carryover of PCR products from one reaction to the next.

Like most mucosal pathogens, E. coli can be said to follow a requisite strategy of infection: (i) colonization of a mucosal site, (ii) evasion of host defenses, (iii) multiplication, and (iv) host damage. The most highly conserved feature of diarrheagenic E. coli strains is their ability to colonize the intestinal mucosal surface despite peristalsis and competition for nutrients by the indigenous flora of the gut (including other E. coli strains). The presence of surface adherence fimbriae is a property of virtually all E. coli strains, including nonpathogenic varieties. However, diarrheagenic E. coli strains possess specific fimbrial antigens that enhance their intestinal colonizing ability and allow adherence to the small bowel mucosa, a site that is not normally colonized (389, 679). The various morphologies of E. coli fimbriae are illustrated in Fig. 2. The role of fimbrial structures in adherence and colonization is often inferred rather than demonstrated, in part due to the host specificity of most fimbrial adhesins.

View larger version (99K): [in this window] [in a new window]   View larger version (103K): [in this window] [in a new window]   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 µm. Reprinted from reference 245 with permission of the publisher.

Once colonization is established, the pathogenetic strategies of the diarrheagenic E. coli strains exhibit remarkable variety. Three general paradigms have been described by which E. coli may cause diarrhea; each is described in detail in the appropriate section below: (i) enterotoxin production (ETEC and EAEC), (ii) invasion (EIEC), and/or (iii) intimate adherence with membrane signalling (EPEC and EHEC). However, the interaction of the organisms with the intestinal mucosa is specific for each category. Schematized paradigms are illustrated in Fig. 3.

View larger version (34K): [in this window] [in a new window]   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.

The versatility of the E. coli genome is conferred mainly by two genetic configurations: virulence-related plasmids and chromosomal pathogenicity islands. All six categories of diarrheagenic E. coli described in this review have been shown to carry at least one virulence-related property upon a plasmid. EIEC, EHEC, EAEC, and EPEC strains typically harbor highly conserved plasmid families, each encoding multiple virulence factors (275, 467, 701). McDaniel and Kaper have shown recently that the chromosomal virulence genes of EPEC and EHEC are organized as a cluster referred to as a pathogenicity island (431, 432). Such islands have been described for uropathogenic E. coli strains (163) and systemic E. coli strains (75) as well and may represent a common way in which the genomes of pathogenic and nonpathogenic E. coli strains diverge genetically. Plasmids and pathogenicity islands carry clusters of virulence traits, yet individual traits may be transposon encoded (such as ST) (607) or phage encoded (such as Shiga toxin) (485).

In the sections that follow, we will review all aspects of disease due to the different classes of diarrheagenic E. coli. Since diarrheagenic E. coli strains are distinguished and defined on the basis of pathogenetic mechanisms, much of this review will concern the latest advances in our knowledge of the pathogenesis of these organisms.

ETEC is defined as containing the E. coli strains that elaborate at least one member of two defined groups of enterotoxins: ST and LT (381). ETEC strains were first recognized as causes of diarrheal disease in piglets, where the disease continues to cause lethal infection in newborn animals (reviewed in reference 15). Studies of ETEC in piglets first elucidated the mechanisms of disease, including the existence of two plasmid-encoded enterotoxins. The first descriptions of ETEC in humans reported that certain E. coli isolates from the stools of children with diarrhea elicited fluid secretion in ligated rabbit intestinal loops (642). DuPont et al. subsequently showed that ETEC strains were able to cause diarrhea in adult volunteers (175).

ETEC strains are generally considered to represent a pathogenic prototype: the organisms colonize the surface of the small bowel mucosa and elaborate their enterotoxins, giving rise to a net secretory state. Some investigators have reported that ETEC strains may exhibit limited invasiveness in cell cultures, but this has not been demonstrated in vivo (189, 190). ETEC strains cause diarrhea through the action of the enterotoxins LT and ST. These strains may express an LT only, an ST only, or both an LT and an ST. These toxins have recently been reviewed (291, 293, 295, 296, 480, 589), and the reader is referred to these sources for primary references.

Heat-labile toxins. The LTs of E. coli are oligomeric toxins that are closely related in structure and function to the cholera enterotoxin (CT) expressed by Vibrio cholerae (596). LT and CT share many characteristics including holotoxin structure, protein sequence (ca. 80% identity), primary receptor identity, enzymatic activity, and activity in animal and cell culture assays; some differences are seen in toxin processing and secretion and in helper T-lymphocyte responses (153). There are two major serogroups of LT, LT-I and LT-II, which do not cross-react immunologically. LT-I is expressed by E. coli strains that are pathogenic for both humans and animals. LT-II is found primarily in animal E. coli isolates and rarely in human isolates, but in neither animals nor humans has it been associated with disease. Unless otherwise distinguished by Roman numerals, the term LT below refers to the LT-I form.

(i) LT-I. LT-I is an oligomeric toxin of ca. 86 kDa composed of one 28-kDa A subunit and five identical 11.5-kDa B subunits (Fig. 4A) (622). The B subunits are arranged in a ring or "doughnut" and bind strongly to the ganglioside GM₁ and weakly to GD1b and some intestinal glycoproteins (643). The A subunit is responsible for the enzymatic activity of the toxin and is proteolytically cleaved to yield A₁ and A₂ peptides joined by a disulfide bond. Two closely related variants of LT-I which exhibit partial antigenic cross-reactivity have been described. These variants are called LTp (LTp-I) and LTh (LTh-I) after their initial discovery in strains isolated from pigs or humans, respectively. The genes encoding LT (elt or etx) reside on plasmids that also may contain genes encoding ST and/or colonization factor antigens (CFAs).

View larger version (33K): [in this window] [in a new window]   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  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 Cl by a direct effect, and HCO3 indirectly) by crypt cells and a decrease in absorption of Na+ and Cl 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).

S

(ii) LT-II. The LT-II serogroup of the LT family shows 55 to 57% identity to LT-I and CT in the A subunit but essentially no homology to LT-I or CT in the B subunits (229, 271, 518, 589, 612). Two antigenic variants, LT-IIa and LT-IIb, which share 71 and 66% identity in the predicted A and B subunits, respectively, have been described. LT-II increases intracellular cAMP levels by similar mechanisms to those involved with LT-I toxicity, but LT-II uses GD1 as its receptor rather than GM₁ (229). As noted above, there is no evidence that LT-II is associated with human or animal disease.

Heat-stable toxins. In contrast to the large, oligomeric LTs, the STs are small, monomeric toxins that contain multiple cysteine residues, whose disulfide bonds account for the heat stability of these toxins. There are two unrelated classes of STs that differ in structure and mechanism of action. Genes for both classes are found predominantly on plasmids, and some ST-encoding genes have been found on transposons. STa (also called ST-I) toxins are produced by ETEC and several other gram-negative bacteria including Yersinia enterocolitica and V. cholerae non-O1. STa has about 50% protein identity to the EAST1 ST of EAEC, which is described further below. It has recently been reported (564, 706) that some strains of ETEC may also express EAST1 in addition to STa. STb has been found only in ETEC.

(i) STa. The mature STa is an 18- or 19-amino-acid peptide with a molecular mass of ca. 2 kDa. There are two variants, designated STp (ST porcine or STIa) and STh (ST human or STIb), after their initial discovery in strains isolated from pigs or humans, respectively. Both variants can be found in human ETEC strains. These two variants are nearly identical in the 13 residues that are necessary and sufficient for enterotoxic activity, and of these 13 residues, 6 are cysteines which form three intramolecular disulfide bonds. STa is initially produced as a 72-amino-acid precursor (pre-pro form) that is cleaved by signal peptidase 1 to a 53-amino-acid peptide (533). This form is transported to the periplasm, where the disulfide bonds are formed by the chromosomally encoded DsbA protein (708). An undefined protease processes the pro-STa to the final 18- or 19-residue mature toxin which is released by diffusion across the outer membrane.

(ii) STb. STb is associated primarily with ETEC strains isolated from pigs, although some human ETEC isolates expressing STb have been reported. STb is initially synthesized as a 71-amino-acid precursor protein, which is processed to a mature 48-amino-acid protein with a molecular weight of 5.1 kDa (23, 171). The STb protein sequence has no homology to that of STa, although it does contain four cysteine residues which form disulfide bonds (23). Unlike STa, STb induces histologic damage in the intestinal epithelium, consisting of loss of villus epithelial cells and partial villus atrophy. The receptor for STb is unknown, although it has been suggested recently that the toxin may bind nonspecifically to the plasma membrane prior to endocytosis (115). Unlike the chloride ion secretion elicited by STa, STb stimulates the secretion of bicarbonate from intestinal cells (589). STb does not stimulate increases in intracellular cAMP or cGMP concentrations, although it does stimulate increases in intracellular calcium levels from extracellular sources (170). STb also stimulates the release of PGE₂ and serotonin, suggesting that the ENS may also be involved in the secretory response to this toxin (228, 294).

Colonization factors. The mechanisms by which ETEC strains adhere to and colonize the intestinal mucosa have been the subject of intensive investigation (for recent reviews, see references 109, 149, 230, and 697). To cause diarrhea, ETEC strains must first adhere to small bowel enterocytes, an event mediated by surface fimbriae (also called pili). Transmission electron microscopy of ETEC strains typically reveals many fimbriae peritrichously arranged around the bacterium; often, multiple fimbrial morphologies can be visualized on the same bacterium (389) (Fig. 2B). A large number of ETEC fimbrial antigens have been characterized (Table 3), although the fimbriae of some ETEC strains have yet to be identified and are only presumed to exist. Clearly, the antigenic heterogeneity conferred by the existence of multiple fimbrial antigens is an obstacle to effective vaccine development.

View larger version (27K): [in this window] [in a new window]   FIG. 5.   Genetics of E. coli fimbriae. Genes required for the expression of functional pili are characteristically linked in gene clusters. The genetic organization of these clusters is illustrated for ETEC fimbriae CS1, CFA/I, CS3, and CS6, and for members of the Dr family, found in DAEC and EAEC. Italicized 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 pattern have genetic and functional homology; black arrows represent structural subunits. The known functions of the genes in the Dr cluster are listed below the corresponding genes. These functions can be extrapolated to arrows of similar fill 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 in pap fimbriae: usher proteins are OMPs which serve as pores for the transport and assembly of the fimbrial shaft; fimbrial chaperones bind to the fimbrial subunit proteins in the periplasmic space and prevent premature folding and degradation. CS1 and CFA/I accessory genes, required for assembly and transport of the fimbriae, are homologous to each other but not to CS3, CS6, or the Dr family. CS6 has an unusual organization in that the first two genes of the cluster apparently encode heterologous major subunit proteins (699); the significance of this feature is not yet understood.

ETEC strains are associated with two major clinical syndromes: weanling diarrhea among children in the developing world, and traveler's diarrhea. The epidemiologic pattern of ETEC disease is determined in large part by a number of factors: (i) mucosal immunity to ETEC infection develops in exposed individuals; (ii) even immune asymptomatic individuals may shed large numbers of virulent ETEC organisms in the stool; and (iii) the infection requires a relatively high infectious dose (175). These three features create a situation in which ETEC contamination of the environment in areas of endemic infection is extremely prevalent, and most infants in such areas will encounter ETEC upon weaning. The percentage of cases of sporadic endemic infant diarrhea which are due to ETEC usually varies from 10 to 30% (12, 209, 298, 385, 406, 581, 654). School-age children and adults typically have a very low incidence of symptomatic ETEC infection. Characteristically, ST-producing ETEC strains cause the majority of endemic cases (12, 385).

Epidemiologic investigations have implicated contaminated food and water as the most common vehicles for ETEC infection (71, 73, 395, 700). Sampling of both food and water sources from areas of endemic infection have demonstrated strikingly high rates of ETEC contamination (550, 700); this is not unexpected given that 10⁸ CFU of ETEC with buffer must be given to induce high attack rates in volunteers (175, 383). Thus, fecal contamination of water and food sources is the principal reason for the high incidence of ETEC infection throughout the developing world, and the institution of appropriate sanitation is the cornerstone of preventive efforts against this infection.

ETEC infections in areas of endemic infection tend to be clustered in warm, wet months, when multiplication of ETEC in food and water is most efficient (381). Person-to-person transmission was not found to occur during a study of ETEC-infected volunteers housed side by side with volunteers enrolled in an evaluation of influenza vaccine candidates (388).

Although ETEC infection occurs most frequently in infants, immunologically naive adults are susceptible (this stands in contrast to EPEC infection, as described below). Indeed, ETEC is the predominant etiologic agent causing traveler's diarrhea among adults from the developed world visiting areas where ETEC infection is endemic (21, 70, 174, 422). Studies suggest that 20 to 60% of such travelers experience diarrhea; typically, 20 to 40% of cases are due to ETEC. Predictably, ETEC traveler's diarrhea occurs most commonly in warm and wet months and among first-time travelers to the developing world (21). Traveler's diarrhea is usually contracted from contaminated food and water (70, 422, 700).

The clinical characteristics of ETEC disease are consistent with the pathogenetic mechanisms described above. Similar features of the illness have been demonstrated in both volunteers and patients in areas of endemic infection. The illness is typically abrupt in onset with a short incubation period (14 to 50 h) (175, 459). The diarrhea is watery, usually without blood, mucus, or pus; fever and vomiting are present in a minority of patients (175, 381). ETEC diarrhea may be mild, brief, and self-limiting or may result in severe purging similar to that seen in V. cholerae infection (383).

Most life-threatening cases of ETEC diarrhea occur in weanling infants in the developing world. Even though the administration of antibiotics to which ETEC strains are susceptible has been shown to decrease both the duration of diarrhea and the intensity of ETEC excretion (72, 173), effective 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 management 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 diarrhea. In addition, bismuth subsalicylate or loperamide is effective 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, norfloxacin, and ofloxacin) are the most commonly recommended agents, since increasing antimicrobial resistance to traditional agents has been documented in several areas (173, 174).

Travelers to developing areas are often concerned with the development of traveler's diarrhea and may seek a means of preventing it. Doxycycline and trimethoprim-sulfamethoxazole have been shown to be effective in this regard, although increasing resistance would suggest that fluoroquinolones administered once daily would be more effective (280). However, the growing problem of antibiotic resistance and the possibility of adverse effects from antimicrobial agents weigh strongly against recommending antimicrobial prophylaxis routinely. Rather, experts have recommended (i) avoiding potentially contaminated food and drink while traveling, (ii) bismuth subsalicylate given four times daily, and (iii) the use of antibiotics empirically if significant diarrhea develops (174).

Oral vaccines against ETEC are being developed by a variety of approaches including the use of killed whole cells, toxoids, purified fimbriae, attenuated ETEC strains, and attenuated Salmonella, Shigella, and V. cholerae strains expressing ETEC antigens (reviewed in references 626 and 630). An oral cholera vaccine containing killed V. cholerae and purified CT B subunit has been reported to provide protection against traveler's diarrhea due to ETEC (511). This protection is presumably due to the antigenic similarity between LT and CT, although this would not explain the protection against ETEC strains expressing ST. Development of an ETEC vaccine with broad protection is greatly complicated by the numerous intestinal colonization factors expressed by ETEC.

Detection of ETEC has long relied on detection of the enterotoxins LT and/or ST. ST was initially detected in a rabbit ligated ileal loop assay (193), but the expense and lack of standardization caused this test to be replaced by the suckling-mouse assay (236), which became the standard test for the presence of STa for many years. The suckling-mouse assay entails the measurement of intestinal fluid in CD4 infant mice after percutaneous injection of culture supernatants.

Several immunoassays have been developed for detection of ST, including a radioimmunoassay (237) and an enzyme-linked immunosorbent assay (ELISA) (144) (available from Denka Seiken, Co. Ltd., Tokyo, Japan). Both of these tests correlate well with results of the suckling-mouse assay and require substantially 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 hamster ovary (CHO) cell assay. In the Y1 assay, ETEC culture supernatants are added to Y1 cells and the cells are examined for rounding (165). In the CHO cell assay, LT will cause elongation of the CHO cells (265). Immunologic assays are easier to implement in clinical laboratories and include the traditional Biken test (297) as well as newer immunologic methods such as ELISA (709), latex agglutination (304), and two commercially available tests, the reversed passive latex agglutination test (582) and the staphylococcal coagglutination test (116). Both of the commercially available tests are reliable and easy to perform (613).

ETEC strains were among the first pathogenic microorganisms for which molecular diagnostic techniques were developed. As early as 1982 (455), DNA probes were found to be useful in the detection of LT- and ST-encoding genes in stool and environmental samples. Since that time, several advances in ETEC detection have been made, but genetic techniques continue to attract the most attention and use. It should be stressed that there is no perfect test for ETEC: detection of colonization factors is impractical because of their great number and heterogeneity; detection of LT and ST defines an ETEC isolate, yet many such isolates will express colonization factors specific for animals and thus lack human pathogenicity.

The LT polynucleotide probe provides good sensitivity and specificity when labeled with radioisotopes (373, 455) or with enzymatic, nonisotopic detection systems (528). Several different protocols have been published in which nonisotopic labeling methods have proven useful for LT detection (2, 117, 718); we now use a highly reliable alkaline phosphatase-based detection system (Blue Gene; Gibco-BRL) for use in polynucleotide probe colony blot hybridization.

ST polynucleotide probes have had problems of poor sensitivity and specificity, presumably because of the small size of the gene. For this reason, oligonucleotide probes which are generally more sensitive and specific for ST detection have been developed (581) (Table 2 lists the nucleotide sequences of oligonucleotides used for probing and PCR of diarrheagenic E. coli strains). An LT oligonucleotide has also been developed (581), but this reagent has relatively few advantages over an enzymatically detected LT fragment probe. Recently, a trivalent oligonucleotide probe has been proposed which may be of use in detecting the genes encoding LT, ST, and the EHEC Shiga toxin genes (see below); this probe shows promise in an early report (44). ETEC strains are particularly amenable to stool blot hybridization because of the large number of organisms typically shed in the stools of infected individuals (615).

Several PCR assays for ETEC are quite sensitive and specific (177, 374, 492, 581, 615, 654) when used directly on clinical samples or on isolated bacterial colonies. A useful adaptation of PCR is the "multiplex" PCR assay (374, 615), in which several PCR primers are combined with the aim of detecting one of several different diarrheagenic E. coli pathotypes in a single reaction. After multiplex PCR, various reaction products can usually be differentiated by product size, but a second detection step (e.g., nonisotopic probe hybridization) is generally performed to identify the respective PCR products definitively.

Attaching-and-effacing histopathology. The hallmark of infections due to EPEC is the attaching-and-effacing (A/E) histopathology, which can be observed in intestinal biopsy specimens from patients or infected animals and can be reproduced in cell culture (18, 314, 358, 453, 524, 547, 616, 640, 667, 669) (Fig. 6). This striking phenotype is characterized by effacement of microvilli and intimate adherence between the bacterium and the epithelial cell membrane. Marked cytoskeletal changes, including accumulation of polymerized actin, are seen directly beneath the adherent bacteria; the bacteria sometimes sit upon a pedestal-like structure. These pedestal structures can extend up to 10 µm out from the epithelial cell in pseudopod-like structures (453). This lesion is quite different from the histopathology seen with ETEC strains and V. cholerae, in which the organisms adhere in a nonintimate fashion without causing microvillous effacement or actin polymerization. Although earlier studies had also reported this histopathology, it was not until the report by Moon et al. (453) that the phenotype became widely associated with EPEC and the term "attaching and effacing" was coined.

View larger version (141K): [in this window] [in a new window]   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.

Three-stage model of EPEC pathogenesis. Multiple steps are involved in producing the characteristic A/E histopathology. In 1992, Donnenberg and Kaper (158) proposed a three-stage model of EPEC pathogenesis consisting of (i) localized adherence, (ii) signal transduction, and (iii) intimate adherence (Fig. 7). The temporal sequence of these stages is not certain, and, indeed, the different stages may occur concurrently. Nevertheless, this model has proven to be a robust one that can readily accommodate advances in our understanding of EPEC pathogenesis that have been made since it was first proposed. Additional details on this model can be found in recent reviews (154, 159, 327).

View larger version (25K): [in this window] [in a new window]   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.

(i) Localized adherence. As noted above, adherence to HEp-2 cells was first described by Cravioto et al. for EPEC (139). Baldini et al. (26) showed that the ability of EPEC strain E2348/69 (O127:H6) to adhere in a localized pattern was dependent on the presence of a 60-MDa plasmid. Loss of this plasmid led to loss of the LA phenotype, and transfer of this plasmid to nonadherent E. coli HB101 enabled this strain to adhere to HEp-2 cells. This plasmid was therefore designated the EPEC adherence factor (EAF) plasmid (see below), and a 1-kb fragment from this region was developed as a diagnostic DNA probe (the EAF probe) (27, 461). Although this probe proved to be extremely valuable in diagnosing EPEC (see below) and elucidating the epidemiology of EPEC infections, the exact nature of the adhesin mediating this adherence remained unknown for many years.