Mechanisms of gut damage by Escherichia coli

3 Mechanisms of gut damage by Escherichia coli A. D. PHILLIPS

BA, PhD Clinical Scientist and Honorary Senior Lecturer

University Department of Paediatric Street, London NW3 ZQG, UK

G. FRANKEL

Gastroenterology,

Third

Floor,

Royal

Free

Hospital,

and Medicine,

London,

Pond

BSc, PhD

Lecturer Department

of Biochemistry,

Imperial

College

of Science,

Technology

UK

This chapter primarily concerns three main categories of diarrhoeagenic Escherichia coli, enteropathogenic (EPEC), enterohaemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. They have distinctive virulence factors and vary in the enteropathies they produce. The molecular biological approach has opened up the complex way in which they interact with the intestine. EPEC and EHEC show a subversive approach to colonization in that they adapt the host cell to their requirements in the formation of the attaching effacing lesion. EAEC appear to co-opt the host defence system to produce a biofilm-like colony and currently go unrecognized in routine laboratories. Key words: Escherichia Escherichia

Escherichiu coli 0157; coli.

infections; enteropathogenic enterohaemorrhagic Escherichia

coli

Escherichia coli;

coli; intimin; enteroaggregative

Escherichia coli comprises several diarrhoeagenic categories, with somewhat diverse pathogenetic properties. Currently five groups of diarrhoeagenie E. coli are recognized: enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohaemorrhagic (EHEC), enteroaggregative (EAEC) and enteroinvasive (EIEC). All groups have the common virulence characteristic of adhesion to the mucosal surface; they differ, however, in their capacity to secrete entero- and cytotoxins, to induce enteropathy and to invade the mucosa (Levine, 1987). For the purposes of this chapter, the first four groups will be considered in turn with regard to their interaction with the gut, the extent of damage they cause and the mechanisms that have been elucidated to date. EIEC will not be considered further in view of limitations of space. Another category of E. coli, termed diffuse adhering E. coli (DAEC) on the basis of their pattern of adherence to HEp-2 cells (Nataro et al 1987), has an uncertain association with human diarrhoeal disease and will be considered only briefly. BailliPre

S Clinical

Gastroenterology-

Vol. 11, No. 3, September 1997 ISBN 0-702%2384-l 0950-3528/97/030465 + 19 $12.00/00

465 Copyright 0 1997, by Bail&e Tindall All rights of reproduction in any form reserved

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ETEC express several surface adhesins (fimbriae) which provide the means of adhering to the mucosal surface and secrete heat labile and stable enterotoxins (Levine, 1987). However, they have not been shown to cause overt mucosal damage in humans and animals (Figure 1). The similarities between cholera toxin and E. coli heat-labile toxin suggest that cytological changes reported in cholera in vivo (Mathan et al, 1995) may be seen in ETEC.

(4

(B) Figure 1. Scanning electron microscopy (EM) of natural ETEC infection (in the weaning pig): (A) view of one villus showing carpet of adhering bacteria (arrow, x 1190); (B) detail of adhering bacteria demonstrating close adherence with no overt damage to the microvillous brush border (X 15 300).

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A recent excellent review of EPEC (Kaper et al, 1996) has been published, making a detailed overview of some areas unnecessary. EPEC is a common cause of diarrhoea, particularly among young infants in developing countries (Levine and Edelman, 1984; Albert et al, 1995). Clinical and epidemiological investigations demonstrate that breast feeding is protective against infectious diarrhoea, including EPEC infection (Blake et al, 1993). The reasons for infantile susceptibility to EPEC infection are unclear. Protection from infection with age may be due to immunity arising from previous exposure and/or due to changes in receptor expression. Certainly, there is evidence of variation in bacterial adhesion and toxin binding with age: in newborn humans and animals increased receptors for bacterial toxins are present compared with adults (Cohen et al, 1988). Symptoms in EPEC infection range from acute self-limiting gastroenteritis to protracted life-threatening diarrhoea (Hill et al, 1991). Traditionally, EPEC is considered to comprise strains of 12 different 0 serogroups: 026, 055,086,0111,0114,0119,0125,0126,0127,0128,0142 and 0158 (World Health Organization, 1987). Population genetic surveys, using multilocus enzyme electrophoresis, have shown that EPEC are divided into two major groups of related clones, designated EPEC 1 and EPEC 2 (Whittam and McGraw, 1996). Within each group, a variety of 0 antigens are present while the flagellar antigen is conserved. Strains belonging to EPEC 1 typically express antigens H6 and H34, whereas EPEC 2 strains express antigen H2. Damage to the gut A characteristic attaching-effacing lesion of the brush border with severe crypt hyperplastic villous atrophy (Figure 2) has been described in the small intestine of children with chronic diarrhoea who are infected with EPEC of both groups 1 and 2 (Ulshen and Rollo, 1980; Rothbaum et al, 1982). Ethical considerations discourage similar studies in children with acute EPEC infections, although an early study by Toccalino et al (1971) showed the presence of an enteropathy, and animal studies using EPEC isolated from children with acute diarrhoea show identical attaching and

Figure chronic

2. Severe diarrhoea

crypt hyperplastic villous and failure to thrive.

atrophy

in an in vivo EPEC

infection

in a child

with

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effacing lesions in association with an enteropathy (Moon et al, 1983). Thus, it is believed that the EPEC-induced lesion in acute gastroenteritis is the same as that produced in chronic diarrhoea. The attaching and effacing lesion is characterized by destruction of microvilli and intimate attachment of bacteria to cup-like pedestals of the apical membrane (Moon et al, 1983). Infection of cultured epithelial cells by EPEC reproduces the attaching and effacing lesion and shows a ‘localized’ pattern of adherence (Scaletsky et al, 1984), similar to that seen in vivo. Attaching and effacing lesions are produced by some other bacteria, including EHEC (Donnenberg et al, 1993) and Citrobacter rodentium (Schauer and Falkow, 1993). The mechanism by which EPEC produce attaching and effacing lesions, severe enteropathy and diarrhoea is unclear, but much progress has been made in determining the factors involved, particularly by adopting a molecular biological approach. The genetic basis of localized effacing lesion

adherence

and the attaching

and

Experiments using cultured epithelial cells have implicated several genes (and their associated proteins) in localized adhesion and attaching and effacing lesion formation; these genes map to two sites. 0

l

One site is a 35 kbp chromosomal locus termed the locus of enterocyte effacement (LEE) region (McDaniel et al, 1995). The LEE region, found in all attaching and effacing lesion forming bacteria (McDaniel et al, 1995), encodes a type III secretion system (Jarvis et al, 1995), a series of secreted effector proteins (EPEC secreted proteins or Esp), including EspA, EspB and EspD (Donnenberg et al, 1993; Kenny et al, 1996; Lai et al, 1997) and intimin, product of the eae gene (Jerse et al, 1990; Frankel et al, 1996b). The other is the 50-70 MDa EPEC adherence factor (EAF) plasmid (Nataro et al, 1985) which encodes bundle-forming pili (Giron et al, 1991a; Donnenberg et al, 1992), and has a regulatory locus (the per locus) that regulates and co-ordinates the expression of several EPEC virulence factors (including intimin and bundle-forming pili) (GomezDuarte and Kaper, 1995; Tobe et al, 1996; Knutton et al, 1997).

The three-stage

model of enteropathogenic

E. coli pathogenesis

Observations on the interaction of EPEC with cell culture systems have produced a three-stage model of EPEC pathogenesis (Donnenberg and Kaper, 1992): (1) bundle-forming pili mediated initial, non-intimate, adherence which brings the bacteria into contact with the epithelial cell surface, (2) Esp-dependent tyrosine-kinase-mediated signal transduction which activates a 90kDa, host cell associated surface protein (Hp90 (Rosenshine et al, 1996)) that acts as a receptor for intimin and (3) intiminmediated close attachment, cytoskeletal rearrangement involving changes in calcium concentrations (Baldwin et al, 1991) and pedestal formation.

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However, the limited information on the interaction of EPEC with the mucosa in humans means that the suitability of the model for natural EPEC infection is not clear. Data produced using in vitro organ culture (IVOC) of paediatric intestine infected with wild-type and specific EPEC mutants have indicated that bundle-forming pili are not responsible for initial nonintimate adherence but may be more involved in later stages within localized adhering colonies (Hicks et al, 1997). An intimin-deficient EPEC (termed CVD206 (Donnenberg and Kaper, 1991)) which expresses bundleforming pili at the wild-type level and adheres to epithelial cells in culture could not be shown to adhere to paediatric intestine in vitro; in contrast, the wild-type EPEC (E2348/69), the eae-complemented CVD206 (termed CVD206 (pCVD438) (Donnenberg and Kaper, 1991)) and JPNl5 (an EAF virulence plasmid cured derivative of EPEC E2348/69) (Jerse et al, 1990) strains all adhered and formed attaching and effacing lesions. Thus initial mucosal adherence and attaching and effacing lesion formation were achieved in the absence of bundle-forming pili. However, JPNl5 did not form typical three-dimensional microcolonies, but the bacteria adhered in a flatter manner (Hicks et al, 1997). These results stress the importance of intimin and indicate that it is essential for binding to human mucosa. Bundle-forming pili are probably needed for subsequent bacterial-bacterial contacts and, in the absence of intimin-mediated attachment, may allow bacteria to separate from colonies and to colonize other areas of the gut (Hicks et al, 1997). It also follows that intimin and/or other adhesin(s) (which await elucidation) are involved in early mucosal adhesion. Because of the central importance of intimin in EPEC pathogenesis, some detailed consideration will be given to its properties. Intimin

cell-binding

domain and its host cell receptor(s)

For intimin to mediate intimate adherence to eukaryotic cells it must bind to cell-surface receptors. The cell-binding activity of intimin has been recently localized to its carboxy-terminal 280 amino acids (In&,) and Cys937 was shown to be essential for this activity (Frankel et al, 1995). The same group showed that purified intimin polypeptide can bind to HEp-2 cells without prior activation by EPEC-secreted proteins (Frankel et al, 1994). This contradicts stage two of the three-stage model (see above) where it is considered that intimin interacts with Hp90 and that this interaction is dependent on prior tyrosine phosphorylation of Hp90 (Rosenshine et al, 1996). Moreover, recently Rabinowitz et al (1996) have shown that attaching and effacing activity of EPEC can still occur in the presence of genistein, a potent tyrosine kinase inhibitor (Rosenshine et al, 1992). Also, attaching and effacing lesion formation without detectable tyrosine-kinasemediated signal transduction was shown in an sepZ EPEC mutant (Rabinowitz et al, 1996) and in 0157:H7 EHEC (Ismaili et al, 1995). There is also some evidence that intimin may bind to integrins (Frankel et al, 1996a). This is not surprising in view of its homology with Yersinia invasin which binds to the a,6p, integrins, heterodimeric glycoproteins that

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transmit signals across the plasma membrane after interaction with a variety of extracellular ligands (Isberg and Leong, 1990). However, direct evidence of intimin interaction with 0, integrins during attaching and effacing adherence is lacking, and further research is needed in this area. Regulation

of surface intimin

expression

Recent work has indicated that surface expression of intimin is regulated during attaching and effacing lesion formation and that virulence plasmidencoded genes participate in this process (Knutton et al, 1997). Surface expression of intimin is readily detected during the early stages of EPEC-host cell interactions. However, intimin expression decreases following attaching and effacing lesion formation. EAF plasmid cured strain JPN15 did not show this decrease, unless it was complemented with plasmid-encoded regulatory (per) genes. The nature of the signals transduced from infected host cells to adherent bacteria and whether intimin expression is regulated at the transcriptional or post-transcriptional level are not known. Intimin derivatives expressed by enteropathogenic enteropathogenic E. coli related strains

E. coli and

Intimin can be divided antigenically into different groups (Agin and Wolf, 1997; Adu-Bobie et al, submitted for publication) which have been termed a, p, ‘y, 6 and E (Adu-Bobie et al, submitted for publication). The first group (intimin a) all belong to EPEC 1; the second group (intimin p) belong to EPEC clone 2 (apart from 0119:H6), C. rodentium and rabbit diarrhoeagenie E. coli (RDEC-1). Intimin y strains belonged to serotypes 055:H-, 055:H7, and to its related EHEC clone, 0157:H7. Intimins S and E were produced by strains 086:H34 and 0127:H40 respectively. Intimin

and cellular-regional

specificity

In humans, EPEC mainly colonizes the small intestine whereas EHEC is found in the colon in animal models (Tzipori et al, 1986, 1989) and there are clear differences in the primary structures of the cell-binding domains of intimin a and y (Yu and Kaper, 1992). In a newborn piglet model when intimin y of EHEC was replaced with intimin a, colonization extended into the small intestine (Donnenberg et al, 1993), indicating that intimin may indeed determine the site of intestinal colonization. Recently, it has been shown that wild-type C. rodentium (expressing intimin p) and C. rodentium expressing EPEC-derived intimin a colonize and produce attaching and effacing lesions in only the descending colon of orally challenged mice. However, although adhesion remained tissue specific the cell specificity of C. rode&urn expressing EPEC-derived intimin a appeared to be subtly altered as there was a more extensive colonization of the crypts, where bacteria could be seen in association with cells which are probably at earlier stages of maturation (Frankel et al,

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1996~). It is not clear whether these different adherence patterns reflect differences in binding specificity of intimin a and intimin fi. In rabbits, RDEC-1 initially binds to Peyer’s patch or follicle-associated epithelium (FAE) before colonizing the surrounding mucosa (Cantey and Inman, 1981). It is not known whether EPEC also colonize the intestine in humans in this fashion, although other bacterial species show an association with FAE, i.e. Yersinia (Grutzkau et al, 1990) and SaZrnoneZZa (Trier, 1991). Most of these studies concern animals and the role of M cells in bacterial pathogenesis in humans is unclear. Pedestal formation Experiments with cultured cells have shown raised levels of free calcium in response to EPEC infection (Baldwin et al, 199 l), which may be induced by the release of inositol phosphates, including IP3 (Foubister et al, 1994). In response to the raised calcium it is proposed that the microvillous actin cores would break down and vesiculation would occur. Adhering bacteria could then approach the apical membrane and intimate attachment (via intimin) would follow (Figure 3). Cytoskeletal components accumulate immediately beneath the bacterium and include actin, myosin light chain, ezrin and tallin (Finlay et al, 1992). This area is raised slightly above the surrounding membrane, producing a pedestal (Figure 3). These events occur within 6 hours of IVOC on paediatric intestine and precede any evidence of villous atrophy. Thus, the generation of an enteropathy in vivo should occur subsequent to attaching and effacing lesion formation and bacterial colonization. Recent work analysing the involvement of calcium in EPEC infection has provided strong evidence that changes in calcium levels do not occur during attaching and effacing lesion formation (Bain et al, 1997). If this is so then alternative explanations for microvillous effacement need to be hypothesized. Enteropathy In natural EPEC infection in children there is a moderate to severe enteropathy (Figure 2) characterized by crypt hyperplasia, an increased crypt cell proliferation rate and a high mitotic index (Savidge et al, 1996). The high turnover rate in a mucosa with severe villous atrophy indicates a high level of epithelial cell loss. It is not clear whether this is due to direct cytotoxic action by EPEC, an increased apoptotic rate induced directly or indirectly by EPEC, or both. Disruption of cell-cell and cell-matrix contacts in the intestine increases apoptosis as does the gluten-sensitive enteropathy of coeliac disease (Moss et al, 1996). Preliminary studies using terminal uridine deoxynucleotidyl nick-end labelling of DNA fragments as a means of identifying cells in apoptosis showed increased labelling in the surface epithelium of in vivo EPEC-infected small intestine. In these cases neutrophil infiltration of the epithelium was also present, disrupting epithelial cell integrity, and the EPEC infection may therefore cause apoptosis indirectly. Intimin-induced cytoskeletal changes (Finlay et al,

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1992) could alter cell-cell and/or cell-matrix interactions to produce apoptosis directly (Hem&ton and Gordon, 1995). This latter phenomenon is termed ‘anoikis’ (Frisch and Francis, 1994). In vitro studies of EPEC infection have demonstrated a decrease in transepithelial electrical resistance (Canil et al, 1993) together with increased permeability to low molecular weight sugars, probably via the paracellular route. This may be a result of phosphorylation of myosin light chain (Spitz et al, 1995) following signal transduction events. A transient increase in short-circuit current has also been reported prior to changes in transepithelial resistance, indicating chloride secretion in response to attaching and effacing lesion formation (Collington et al, in press), which may contribute to diarrhoeal symptoms. The reports of the failure of oral rehydration therapy in EPEC infection (Marin et al, 1985) may be related to the high level of crypt cell production as this could result in relatively immature cells appearing in the surface epithelium that are ill equipped to perform absorptive functions. In addition EPEC can colonize large (Figure 3(C)) as well as small bowel (Rothbaum et al, 1982) and this is another potential factor in the failure of oral rehydration therapy. In contrast, oral rehydration therapy in rotavirus infection, despite the finding of a severe enteropathy (Davidson and Barnes, 1979), is generally successful. This is probably due to the patchy and proximal small-intestinal localization of the lesion. Invasion

and enteropathogenic

E. coli

There are isolated case reports of invasive EPEC being found in smallintestinal biopsies from children with diarrhoea (Fagundes Neto et al, 1995). EPEC can be shown to invade HEp-2 cells (Donnenberg et al, 1989). However, in the majority of in vivo cases, invasion by EPEC has not been described, and the relevance of the in vitro evidence to the in vivo situation is unclear. As can be seen, although progress has been made, the subject of EPEC pathogenesis is in a state of some flux regarding the interaction of EPEC

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Figure 3. In viva attaching-effacing lesion of EPEC in children: (A) proximal small intestine showing effacement of microvilli at sites of attachment with pedestal formation (arrow) (transmission EM, x2.5 500); (B) scanning EM appearance, note microvillous elongation at edge of bacterial colony (arrow) (x 10 800); (C) attaching-effacing lesion in the colon (transmission EM, x 13 300).

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and the host. Currently, the involvement of bundle-forming pili, the requirement for signal transduction of host cell proteins via tyrosine kinase in attaching and effacing lesion formation by EPEC in humans and the role of calcium all require careful consideration. Part of the reason for this is the limited amount of in vivo human data which are available.

ENTEROHAEMORRHAGIC

E. COLZ

EHEC, first observed in 1982 (Riley et al, 1983), are also referred to as verotoxigenic E. coli (VTEC) and shigatoxigenic E. cob (STEC). The terms are synonymous. Recent reviews cover many salient features of this important category of diarrhoeagenic E. coli (Whipp et al, 1994; Cohen, 1996; Noel and Boedeker, 1996; Arbus, 1997). The most common serotype reported varies from country to country, and it is important to realize that there are many EHEC serotypes which can cause human disease. In the USA, 0157:H7 is most common (Griffin and Tauxe, 1991), but in Australia 011 l:H- is the most prevalent organism. 026:Hll is half as common as 0157:H7 in the USA but it is twice as common in Germany. EHEC infections are reported world-wide, including the UK, Japan, Africa and South America (Waters et al, 1994; Germani et al, 1997). The organisms are mainly food borne, but person-to-person spread and water-borne contamination are other routes. Ground beef is a common mode of transmission leading to the name of hamburger disease for 0157:H7 infections in the USA, where there have been around 140 outbreaks since the 198Os, involving up to 3000 cases. Other foods involved include unpasteurized apple juice, salads and fruit items (Easton, 1997). The infectious dose is low, calculated to be from 10 to 100 organisms (Boyce et al, 1995). and cases peak in the summer months. Cases present with acute diarrhoea which is often bloody, but without fever, hence the term EHEC. All ages are affected, but the very young and the aged are particularly at risk (Brotman et al, 1995). A serious complication of EHEC infection is the haemolytic uraemic syndrome (Bitzan et al, 1993; Pickering et al, 1994; Cohen, 1996; Arbus, 1997) (see below). The serious nature of the illness and the recent outbreak in Scotland have prompted urgent calls in the UK for more research into EHEC and its transmission. Routine microbiology laboratories, although generally not attempting to identify EPEC, routinely screen for 0157:H7.

Detection EHEC should be suspected in cases of severe diarrhoea, particularly if bloody. They should be looked for in cases of haemolytic uraemic syndrome, and post-diarrhoeal TTP. The bacteria can be grown on sorbitol McConkey agar and serotyping for 0157:H7, or the more common strains, should be performed. However, sorbitol non-fermenting and fermenting strains are now reported (Karch et al, 1993) and non-0157 outbreaks have

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included 026:Hll, 0103:H2,0?:H19,Olll:H-, 0104:H22 and 0145:H-. Enrichment using selective culture media and immunomagnetic separation will aid diagnosis, Shiga toxin production can be looked for using Vero cell cultures (high sensitivity but cumbersome) or an enzyme-linked immunosorbent assay system (easy and fast, broad spectrum). Other forms of typing include phage typing, restriction fragment polymorphism analysis and pulsed-field gel electrophoresis. Pathogenesis EHEC can be divided into strains which are eae intimin gene positive and cause an attaching effacing lesion (as in EPEC) and those which are eae gene negative, where pathogenetic mechanisms are unclear. The bacteria colonize mainly the colon (Griffin et al, 1990) and do not appear to infect the proximal small intestine. Strains are bundle-forming pili negative and research is needed to determine which adhesins are important. Adhesion to HEp-2 cells is poor and 6-hour assays are used to demonstrate localized adhesion, which is attaching-effacing lesion positive in eae gene positive strains. Eae gene negative strains also adhere to HEp-2 cells, but they do not form attaching effacing lesions. All strains, by definition, secrete Shiga toxin. There are two main toxins, types I and II (Acheson et al, 1991), and these can inhibit protein synthesis, induce paralysis and cause microvascular lesions (O’Brien and Holmes, 1987; Richardson et al, 1988). Cytotoxicity is enhanced by the presence of pro-inflammatory cytokines (tumour necrosis factor a and interleukin-1B) (Obrig et al, 198.5). The receptor for Shiga toxin is globotriaosylceramide (GB,) (Jacewicz et al, 1986). Haemolytic

uraemic syndrome

The haemolytic uraemic syndrome (Bitzan et al, 1993; Pickering et al, 1994; Cohen, 1996; Arbus, 1997) is one of the more common causes of acute renal failure in paediatrics world-wide. It is considered to be due to the transfer of the toxin across the intestine into the circulation where it initiates thrombotic microangiopathy at distant sites, including the kidney. Approximately 6% of infections may develop this syndrome and a tenth of these may prove fatal. For example, in an E. coli 0157:H7 multistate outbreak in the USA associated with eating hamburgers (Bell et al, 1994), 501 cases of 0157-associated diarrhoea were reported, 15 1 (3 1%) were hospitalized, 45 (9%) developed haemolytic uraemic syndrome and three (0.6%) died. A high level of complications have been reported in children with haemolytic uraemic syndrome, including pancreatitis, colonic necrosis, glucose intolerance, myocardial dysfunction and pericardial effusions (Brandt et al, 1994). EHEC have emerged as important pathogens because of the potentially severe nature of the disease, both intestinal and extraintestinal, and because of the food-related mode of transmission in a mass market with high consumer demand. This has been clearly recognized and improved food

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hygiene practice and quick clinical recognition of cases should bring better control of this organism. ENTEROAGGREGATIVE

E. COLI

EAEC were first recognized by their distinctive pattern of adhesion to HEp-2 cells, where, they form aggregates in a stacked brick pattern (Nataro et al, 1987). Epidemiological evidence implicated EAEC in acute and chronic diarrhoea in developing countries (Bhan et al, 1989; Cravioto et al, 1991), and there are reports of their detection in developed countries (Cravioto et al, 1979; Chan et al, 1994) and in acquired immunodeficiency syndrome patients (Mayer and Wanke, 1995). EAEC contain a plasmid which is considered to contain diarrhoeagenic virulence factors, but pathogenetic mechanisms remain unclear. Two aggregative adherence fimbriae (AAF) have been identified, AAF-I (Nataro et al, 1992) and AAF-II (Czeczulin et al, 1997), and it is likely that more await discovery (Nataro, 1996). Toxins

Several toxins have been described in EAEC. Enteroaggregative heatstable-like enterotoxin (EAST) has been reported (Savarino et al, 1991, 1993) and has also been found in other bacterial species (Savarino et al, 1996). However, an EAST-positive EAEC did not produce diarrhoea in adult volunteers (Nataro et al, 1995), making its association with diarrhoeal disease unclear. Baldwin et al (1992) described the presence of a heat-labile toxin which is antigenically similar to E. coli haemolysin, and Eslava et al (1993) have reported a 108 kDa protein with toxigenic activity in rat ileal loops. Recently, IVOC has been used to demonstrate an EAEC-associated cytotoxic effect on paediatric colon (Figure 4) (Hicks et al, 1996; Nataro et al; 1996). In particular, it was shown that strain 042, which causes diarrhoea in adult volunteers (Nataro et al, 1995) and is AAF-II positive, adhered strongly to the colonic surface in association with increased epithelial cell extrusion, dilated crypt openings, prominent intercrypt crevices, increased mucus discharge and microvillous vesiculation (Nataro et al, 1996). It also produces 108 kDa protein (Nataro, 1996). Changes in calcium concentration have been demonstrated in HEp-2 cells infected with 042 (Baldwin et al, 1992), and this may be the basis of the microvillous changes. Plasmid-cured 042 showed reduced adhesion and no cytotoxic effects, indicating that plasmid-encoded factors are necessary for the expression of the cytotoxic effect (Nataro et al, 1996). Enteropathy

EAEC-related damage to intestinal mucosa has been shown in several studies. The degree of damage is not established. Initially Vial et al (1988) showed marked damage in ligated loops of rabbit and rat intestine, but

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Figure 4. Scanning EM of in vitro cytotoxic effect of EAEC on paediatric colonic mucosa in organ culture: (A) normal appearance after 8 hours incubation with non-pathogenic strain of E. coli (x 360): (B) increased epithelial cell extrusion, widened intercrypt crevices, opened crypt lumina and adhering clumps of bacteria (arrow) following 8 hours incubation with wild-type EAEC strain isolated from a child with diarrhoea (x 360).

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Tzipori et al (1992) found much milder changes in a gnotobiotic piglet model. Ileal damage has been shown in post-mortem studies of children dying in an EAEC outbreak in Mexico (Eslava et al, 1993), although the mucosal presence of bacteria was not reported. Mild crypt hyperplastic villous atrophy has been demonstrated on proximal small-intestinal biopsies from three children with aggregative E. coli in the stool (based on Hep-2 cell assay); two cases also showed aggregates of bacteria adhering to the mucosal surface (Hicks et al, 1994). All cases were suffering from chronic diarrhoea and failure to thrive. Hicks et al (1996) have demonstrated using IVOC that EAEC can adhere to proximal small intestine, but little damage was seen in the 8-hour incubation period. It appears possible therefore that EAEC may cause small- and large-intestinal damage, but there is more colonization of the distal small intestine and colon (Hicks et al, 1996). Mucus may be important in colonization as EAEC promote mucus release and the bacteria can bind both to mucus and to the mucosal surface (Wanke et al, 1990; Hicks et al, 1996), although adhesion to mucus may form part of the host defence system and would be a way of removing bacteria from the lumen. However, combining mucosal with mucus adhesion could produce an anchored colony which would be wrapped in a protective sheath of mucus. This could be a factor in promoting persistent colonization. EAEC, although associated with mild histological changes and being of variable virulence (Nataro, 1996), are important because of their worldwide occurrence (Cravioto et al, 1979, 1991; Bhan et al, 1989; Chan et al, 1994; Mayer and Wanke, 1995) and their ability to persist in the gut. In addition they would not be recognized in a routine microbiology laboratory unless a HEp-2 assay (Nataro et al, 1987) or a DNA probe test was used. Thus, there is a need for a simple diagnostic test for this organism.

DIFFUSE

ADHERING

E. COLZ

This category of E. coli has been associated with diarrhoea in children in some (Giron et al, 1991b; Poitrineau et al, 1995), but not all (Forestier et al, 1996), epidemiological studies. However, the pathogenic potential of DAEC could not be demonstrated in experimentally challenged adult human volunteers (Tacket et al, 1990). This group of potentially diarrhoeagenic E. coli is highly heterologous (Poitrineau et al, 1995; Forestier et al, 1996), a fact which may be responsible for the discrepant results concerning their involvement in disease. Mucosal damage in humans has not been reported. Acknowledgements Figure 1 came from collaborative studies with Dr Bevis Miller (Department Langford House, Bristol). I am grateful to Dr Susan Hicks (University Gastroenterology, Royal Free Hospital, London) for providing Figure 4.

of Veterinary Medicine, Department of Paediatric

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