Background: Microvillous atrophy, a disorder of intractable diarrhoea in infancy, is characterised by the intestinal epithelial cell abnormalities of abnormal accumulation of periodic acid-Schiff (PAS) positive secretory granules within the apical cytoplasm and the presence of microvillous inclusions. The identity of the PAS positive material is not known, and the aim of this paper was to further investigate its composition.Methods: Formaldehyde fixed sections were stained with alcian blue/PAS to identify the acidic or neutral nature of the material, phenylhydrazine blocking was employed to stain specifically for sialic acid, and saponification determined the presence of sialic acid acetylation. The specificity of sialic acid staining was tested by digestion with mild sulphuric acid. Expression of blood group related antigens was tested immunochemically.Results: Alcian blue/PAS staining identified a closely apposed layer of acidic material on the otherwise neutral ( PAS positive) brush border in controls. In microvillous atrophy, a triple layer was seen with an outer acidic layer, an unstained brush border region, and accumulation within the epithelium of a neutral glycosubstance that contained acetylated sialic acid. Blood group antigens were detected on the brush border, in mucus, and within goblet cells in controls. In microvillous atrophy they were additionally expressed within the apical cytoplasm of epithelial cells mirroring the PAS abnormality. Immuno electron microscopy localised expression to secretory granules.Conclusions: A neutral, blood group antigen positive, glycosubstance that contains acetylated sialic acid accumulates in the epithelium in microvillous atrophy. Previous studies have demonstrated that the direct and indirect constitutive pathways are intact in this disorder and it is speculated that the abnormal staining pattern reflects accumulation of glycocalyx related material.

The value set on behavioral variation for its own sake (or the need to be different) is a personality variable nested within social learning theory; a strong need to be different should, in theory, predispose the individual toward originality (including creativity). A self-report inventory measuring this need value (the vDiffer scale; Joy, 1998) motive was administered to undergraduates who also completed the 16 Personality Factor inventory (16PF; Cattell, Cattell, & Cattell, 1993) and House-Tree-Person (H-T-P; Buck, 1948) drawings. Three advanced art therapy graduate students rated the H-T-P protocols for technical proficiency, creativity, and personal adjustment. The need to be different correlated strongly with four primary traits: positively with Q1 (Openness to Change) and M (Abstractedness) and negatively with G (Rule-Consciousness) and Q3 (Perfectionism). Several other moderate correlations also emerged. Judgments of the technical proficiency and creativity of H-T-P drawings correlated significantly with the vDiffer score. The need to be different significantly predicted the quality of these projective drawings even when the influence of intelligence (16PF Scale B) was controlled for. The need to be different is associated with a distinctive pattern of traits and higher quality artistic expression.

Enteroaggregative Escherichia coli (EAEC) is a diarrheal pathogen defined by its characteristic aggregative adherence (AA) to HEp-2 cells in culture. We have previously shown that EAEC strains secrete a 10-kDa protein that is immunogenic in a human EAEC challenge model. We report here that this protein is encoded by a gene (called aap) lying immediately upstream of that encoding the AggR transcriptional activator, and that aap is under AggR control. The product of aap has a typical signal sequence and is secreted to the extracellular milieu, where it remains noncovalently attached to the surface of the bacterium. EAEC aap mutants aggregate more intensely than the wild-type parent in a number of assays, forming larger aggregates and fewer individual bacteria. Infection of colonic biopsies with wild-type EAEC strain 042 and its aap mutant revealed more dramatic autoagglutination of the mutant compared with the wild-type parent. Our data suggest that the aap gene product participates in formation of a surface coat that acts to disperse the bacteria, thus partially counteracting aggregation mediated by aggregative adherence fimbriae. We have therefore named the aap gene product "dispersin," and we propose that it may be representative of a functional class of colonization factors. Since dispersin is expressed in vivo, is highly immunogenic, and is present in most EAEC strains, it holds considerable promise as an EAEC immunogen.

Enteroaggregative Escherichia coli (EAEC) forms thick biofilms on the intestinal mucosa. Here, we show that most EAEC strains form a biofilm on glass or plastic surfaces when grown in cell culture medium with high sugar and osmolarity. Biofilm-forming ability in two prototype EAEC strains required aggregative adherence fimbriae (AAF), although many other EAEC strains that do not express AAF also developed biofilms under these conditions. Ten thousand transposon mutants of EAEC strain 042 were isolated, and 100 were found to be deficient in biofilm formation. Of these, 93 were either deficient in in vitro growth or mapped to genes known to be required for AAF/II expression. Of the seven remaining insertions, five mapped to one of two unsuspected loci. Two insertions involved the E. coli chromosomal fis gene, a DNA-binding protein that is involved in growth phase-dependent regulation. Using reverse transcription-polymerase chain reaction (RT-PCR), we determined that the effect of fis was at the level of transcription of the AAF/II activator aggR. Biofilm formation also required the product of the yafK gene, which is predicted to encode a secreted 28 kDa protein. The yafK product is required for transcription of AAF/II-encoding genes. Our data do not suggest a role for type 1 fimbriae or motility in biofilm formation. EAEC appears to form a novel biofilm, which may be mediated solely by AAF and may reflect its interactions with the intestinal mucosa.

BACKGROUND EnterohaemorrhagicEscherichia coli (EHEC) constitute a significant risk to human health worldwide, and infections, particularly with serogroup O157:H7, are associated with consumption of a variety of food and water vehicles, particularly food of bovine origin. EHEC cause acute gastroenteritis, bloody diarrhoea, and haemorrhagic colitis; up to 10% of cases develop severe complications, including the haemolytic uraemic syndrome, with a 5% case fatality. A virulence characteristic of enteropathogenic E coli, the attaching/effacing lesion, is considered to be important in EHEC. However, although EHEC produce this lesion on cultured human cells, this has not been demonstrated on human intestinal mucosal surfaces. In addition, the initial site(s) of colonisation of EHEC in humans is not known. AIMS To assess the association of EHEC O157:H7 with paediatric and bovine intestine using in vitro organ culture and determine if attaching/effacing lesions occur. METHODS Ultrastructural analysis of in vitro intestinal organ cultures of human small and large intestine was used to investigate adhesion of O157:H7 EHEC to intestinal surfaces. Bovine intestinal organ culture was used to examine the pathology produced by the same EHEC strain in cattle. RESULTS The study showed that EHEC O157:H7 adhered to human intestinal mucosa. Binding and attaching/effacing lesion formation of O157:H7 in humans was restricted to follicle associated epithelium of Peyer's patches. The same strain caused attaching/effacing lesions on bovine mucosa. CONCLUSIONS O157:H7 targets follicle associated epithelium in humans where it causes attaching/effacing lesions. The same human isolate can cause attaching/effacing lesions in cattle, indicating that similar pathogenic mechanisms operate across human and bovine species

Adhesion to cultured epithelial cells by enteropathogenic Escherichia coli (EPEC) is associated with extensive rearrangement of the host cell cytoskeleton. Evidence has been presented that EPEC adhesion is associated with activation of signal transduction pathways leading to production of a characteristic histopathological feature known as the attaching and effacing (A/E) lesion. A/E lesion formation requires intimin, an EPEC adhesion molecule and several EPEC secreted proteins (EspA, B, D and Tir) involved in cell signalling and protein translocation. In this study it is shown that HEp-2 cells respond during the early stages of infection with two wild-type EPEC strains (B171 and E2348/69) by producing microvillus-like processes (MLP) at the site of initial bacterial adherence. Intimin appears to play a key role in MLP elongation. At later stages of infection with these wild-type EPEC strains, when A/E lesions have formed, the MLP were reduced in number and length to appear as at time zero, and the cell surface in the vicinity of bacterial clusters appeared unaffected. In contrast, infection with EspA- or EspB-negative, but intimin-positive, EPEC strains (UMD872 and UMD864, respectively) resulted in enhanced MLP proliferation and formation of 'cage-like' structures engulfing the bacteria. Inoculating HEp-2 cells with intimin-coated latex spheres induced similar 'cage-like' structures. Caco-2 cells did not show intimin-induced microvillus elongation in response to EPEC infection, although microvillus effacement and reduction in number occurred. Similar phenomena appeared on B171 and E2348/69 infection of paediatric intestine using in vitro organ culture, i.e. elongated microvilli were seen in association with small colonies and at the periphery of large localized colonies, along with evidence of microvillus breakdown and debris in the colony centre. These results show that intimin activates signal transduction pathways involved in the remodelling of the eukaryotic cell surface, probably via binding to a receptor encoded by the host cell.

ABSTRACT Enteroaggregative Escherichia coli (EAEC) strains have been shown to adhere to human intestinal tissue in an in vitro organ culture (IVOC) model, and certain strains manifest mucosal toxicity. We have recently described the EAEC plasmid-encoded toxin (Pet), a member of a specific serine protease subclass of the autotransporter proteins. When injected into rat ileal loops, Pet both elicited fluid accumulation and had cytotoxic effects on the mucosa. Furthermore, the Pet protein caused rises in short circuit current from rat jejunal tissue mounted in a Ussing chamber and rounding of intestinal epithelial cells in culture. We therefore hypothesized that the mucosal pathology induced by EAEC strains in the IVOC model was related to expression of the Pet protein. Here, we have examined the effects of EAEC strain 042 and its isogenic pet mutant in the IVOC model. 042-infected colonic explants exhibited dilation of crypt openings, increased cell rounding, development of prominent intercrypt crevices, and absence of apical mucus plugs. Colonic tissue incubated with the pet mutant exhibited significantly fewer mucosal abnormalities both subjectively and as quantitated morphometrically by measurement of crypt aperture diameter. Mucosal effects were restored upon complementation of the pet mutation in trans . Interestingly, we found that the ability of 042 to damage T84 cells was not dependent upon Pet. The data suggest that the Pet toxin is active on the human intestinal mucosa but that EAEC may have other mechanisms of eliciting mucosal damage.

Journal of Pediatric Gastroenterology and NutritionVolume 28, Issue 5 p. 565-565 E S P G H A N 32nd Annual Meeting; Warsaw, Poland, June 2-5, 1999 INITIAL COLONISATION OF ENTEROHAEMORRHAGIC E COLI O157:H7 AND ENTEROPATHOGENIC E COLI O55 IS TO PEYER'S PATCHES IN MAN A D Phillips, A D Phillips University Department of Paediatric Gastroenterology, Royal Free Hospital, Imperial College, LondonSearch for more papers by this authorS Navabpour, S Navabpour University Department of Paediatric Gastroenterology, Royal Free Hospital, Imperial College, LondonSearch for more papers by this authorS Hicks, S Hicks University Department of Paediatric Gastroenterology, Royal Free Hospital, Imperial College, LondonSearch for more papers by this authorG Frankel, G Frankel Biochemistry Department, Imperial College, LondonSearch for more papers by this author A D Phillips, A D Phillips University Department of Paediatric Gastroenterology, Royal Free Hospital, Imperial College, LondonSearch for more papers by this authorS Navabpour, S Navabpour University Department of Paediatric Gastroenterology, Royal Free Hospital, Imperial College, LondonSearch for more papers by this authorS Hicks, S Hicks University Department of Paediatric Gastroenterology, Royal Free Hospital, Imperial College, LondonSearch for more papers by this authorG Frankel, G Frankel Biochemistry Department, Imperial College, LondonSearch for more papers by this author First published: 01 May 1999 https://doi.org/10.1002/j.1536-4801.1999.tb02251.xRead the full textAbout ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume28, Issue5May 1999Pages 565-565 RelatedInformation

Journal of Pediatric Gastroenterology and NutritionVolume 28, Issue 5 p. 557-557 E S P G H A N 32nd Annual Meeting; Warsaw, Poland, June 2-5, 1999 EFFECT OF ENTEROAGGREGATIVE ESCHERICHIA COLI PLASMID-ENCODED TOXIN [PET] ON HUMAN INTESTINE S Hicks, S Hicks University Dept of Paediatric Gastroenterology, Royal Free Hospital, London, UKSearch for more papers by this authorI R Henderson, I R Henderson University of Maryland School of Medicine, Baltimore, USASearch for more papers by this authorF Navarro-Garcia, F Navarro-Garcia University of Maryland School of Medicine, Baltimore, USASearch for more papers by this authorJ P Nataro, J P Nataro University of Maryland School of Medicine, Baltimore, USASearch for more papers by this authorA D Phillips, A D Phillips University Dept of Paediatric Gastroenterology, Royal Free Hospital, London, UKSearch for more papers by this author S Hicks, S Hicks University Dept of Paediatric Gastroenterology, Royal Free Hospital, London, UKSearch for more papers by this authorI R Henderson, I R Henderson University of Maryland School of Medicine, Baltimore, USASearch for more papers by this authorF Navarro-Garcia, F Navarro-Garcia University of Maryland School of Medicine, Baltimore, USASearch for more papers by this authorJ P Nataro, J P Nataro University of Maryland School of Medicine, Baltimore, USASearch for more papers by this authorA D Phillips, A D Phillips University Dept of Paediatric Gastroenterology, Royal Free Hospital, London, UKSearch for more papers by this author First published: 01 May 1999 https://doi.org/10.1002/j.1536-4801.1999.tb02228.xRead the full textAbout ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume28, Issue5May 1999Pages 557-557 RelatedInformation

Intimins, encoded by eae genes, are outer membrane proteins involved in attaching–effacing (A/E) lesion formation and host cell invasion by pathogenic bacteria, including enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium. A series of intimins, harbouring specific mutations close to the C‐terminus, were constructed using pCVD438, which encodes the eae gene from EPEC strain E2348/69. These mutant plasmids were introduced into EPEC strain CVD206 and C. rodentium strain DBS255, which both contain deletion mutations in their eae genes. CVD206, CVD206(pCVD438) and CVD206(pCVD438) derivatives were assessed for their ability to promote A/E lesion formation or invasion of HEp‐2 cells and to induce A/E lesions on fresh human intestinal in vitro organ cultures (IVOC). The pathogenicity of C. rodentium DBS255 harbouring these plasmid derivatives was also studied in mice. Here, we report that intimin‐mediated A/E lesion formation can be segregated from intimin‐mediated HEp‐2 cell invasion. Moreover, adherence to IVOC, EPEC‐induced microvillus elongation and colonization of the murine intestine by C. rodentium were also modulated by the modified intimins.

ABSTRACT Attaching and effacing (A/E) lesion formation is central to enteropathogenic Escherichia coli (EPEC) pathogenesis. In vitro experiments with human epithelial cell lines have implicated virulence plasmid-encoded bundle-forming pili (BFP) in initial binding and intimin in intimate attachment and A/E lesion formation. This study investigated the role of BFP and intimin in EPEC interactions with pediatric small intestinal biopsy tissue in in vitro organ culture. Organ culture infections (2 to 8 h) were performed with E2348/69 (a wild-type EPEC O127:H6 clinical isolate) and E2348/69 derivatives including CVD206 ( eae deficient), CVD206(pCVD438) ( eae -complemented CVD206), CVD206(pCVD438/01) (expressing intimin, which is nonfunctional due to a single amino acid substitution), JPN15 (spontaneous EPEC adherence factor virulence plasmid-cured E2348/69), and 31-6-1(1) (E2348/69 with a Tn phoA insertion inactivation mutation in the virulence plasmid-encoded bfpA gene). Scanning and transmission electron microscopy revealed that after 8 h E2348/69 and CVD206(pCVD438) (both Int + BFP + ) adhered to all specimens, causing A/E lesions with surrounding microvillous elongation. JPN15 and 31-6-1(1) (both Int + BFP − ) adhered and caused A/E lesions although bacteria adhered in “flat,” two-dimensional groups. CVD206 and CVD206(pCVD438/01) (both Int − BFP + ) did not adhere to any sample, and no pathological tissue changes were seen. Thus, in human intestinal organ culture, BFP do not appear to be involved in the initial stages of EPEC nonintimate adhesion but are implicated in the formation of complex, three-dimensional colonies via bacterium-bacterium interactions. Intimin appears to play an essential role in establishing colonization of EPEC on pediatric small intestinal tissue.

Enteroaggregative Escherichia coli (EAEC) has been implicated as an agent of pediatric diarrhea in the developing world. We have shown previously that EAEC adheres to HEp-2 cells by virtue of a plasmid-encoded fimbrial adhesin designated aggregative adherence fimbria I (AAF/I), the genes for which have been cloned and sequenced. However, not all EAEC strains express AAF/I. Using TnphoA mutagenesis, we have characterized a novel fimbria (designated AAF/II) which mediates HEp-2 adherence of the human-pathogenic strain 042. AAF/II is 5 nm in diameter and does not bind AAF/I antiserum, as determined by immunogold transmission electron microscopy. TnphoA identified a gene (designated aafA) which bears significant homology to aggA, the fimbrial subunit of AAF/I (25% identity and 47% similarity at the amino acid level). When hyperexpressed and purified by polyhistidine tagging, the AafA protein assembled into 5-nm-diameter filaments which bound anti-AAF/II antiserum. The cloned aafA gene complemented a mutation in the aggA gene to confer fimbrial expression from the AAF/I gene cluster, manifesting phenotypes characteristic of AAF/II but not AAF/I. The aafA mutant did not adhere to human intestinal tissue in culture, suggesting a role for AAF/II in intestinal colonization. By using DNA probes for AAF/I and AAF/II derived from fimbrial biosynthesis genes, we show that AAF/I and AAF/II are each found in only a minority of EAEC strains, suggesting that still more EAEC adhesins exist. Our data suggest that AAF adhesins represent a new family of fimbrial adhesins which mediate aggregative adherence in EAEC.

Introduction Attaching/Effacing (A/E) lesion formation is central to EPEC pathogenesis. Experiments with a human epithelial cell-line (HEp-2) in vitro have implicated bundle-forming pili (BFP) in initial binding, and intimin (94kDa outer membrane protein encoded by the chromosomal eaeA gene) in intimate attachment and A/E lesion formation; cell invasion by EPEC is also seen. This study investigated the role of these proteins in EPEC interactions with HEp-2 cells and paediatric small intestinal biopsy tissue using in vitro organ culture. Materials & Methods The bacterial strains used were - E69 (a wild-type EPEC), CVD206 (eaeA deficient), CVD438 (CVD206 complemented with eaeA), and CVD438CS (expressing inactive intimin due to a single amino acid substitution). A 3 hour HEp-2 cell assay was used and 6-8 hour organ culture was performed with tissue from 5 children aged 4-120 months. Each strain was examined at least three times. Scanning EM was used to assess the results. Results HEp-2 cells: E69 and CVD438 (both Int+ BFP+) caused A/E lesions and invasion was seen. CVD206 and CVD438CS (both Int+ BFP+) adhered, but no A/E lesions and no invasion were identified. Small intestine: E69 and CVD438 adhered to all specimens causing A/E lesions with surrounding microvillous elongation; invasion was not apparent. CVD206 and CVD438CS did not adhere to any sample and pathological changes were not seen. Conclusions The differences between HEp-2 cells and paediatric intestine in response to EPEC infection raise questions concerning the role of BFP in A/E lesion formation and of invasion in EPEC pathogenesis in man. Intimin appears to play an essential role in establishing and maintaining colonisation of EPEC on paediatric small intestine.

Introduction EPEC cause attaching/effacing (A/E) lesions on cultured epithelial cells and human intestine. A multi-step process of adhesion, signal transduction, cell membrane & cytoskeletal changes, and intimate attachment with pedestal formation has been proposed. This study looks at the mechanisms whereby EPEC alter host cell surface morphology. Materials & Methods The bacterial strains used were - E69 (wild-type EPEC), CVD206 (eaeA deficient, lacking intimin - essential for intimate attachment), UMD864 (espB deficient, lacking EspB - involved in signal transduction). Strains were incubated with HEp-2 cells for 1,2,3 & 6 hrs and with Caco-2 cells for 3 hrs. Results were analysed by scanning EM. Results HEp-2 cells: E69 - bacterial adhesion produced elongation of microvillous-like surface processes [MLP] at 1-2 hrs; A/E lesions were seen at 3 hrs and MLP were shorter and associated with microvesicles; at 6 hrs cells were covered with bacteria. CVD206 - bacterial adhesion increased from 1-3 hrs and was reduced at 6 hrs; short MLP were present at all times. UMD864 - MLP were elongated around adhering bacteria, becoming more complex with time and enmeshing bacteria in cage-like structures at 3 hrs, MLP were unchanged at other cell sites; at 6 hrs less bacteria were seen but MLP complexes remained ± bacteria. Caco-2 cells: E69 caused A/E lesions with associated elongation, microvesiculation and loss of microvilli; CVD206 produced some microvillous elongation and microvesiculation but no A/E lesions. UMD864 adhesion was not associated with overt microvillous changes or with A/E lesions. Conclusions The early stages of EPEC infection involve intimin-induced elongation of MLP at sites of bacterial attachment which are degraded by EspB. Increasing colonisation of cells in culture requires A/E lesion formation. Complex microvillous elongation was not induced on Caco-2 cells, although EspB-related microvillous degradation was seen, indicating that host-cell type influences responses to EPEC virulence factors despite the similar eventual development of A/E lesion formation.

Enteroaggregative Escherichia coli (EAEC) is an important cause of persistent diarrhea in many developing parts of the world, yet the pathogenetic mechanisms of EAEC diarrhea are unknown. Experiments with animal models suggest that EAEC strains damage the intestinal mucosa, and a putative cytotoxin has been described. To characterize the mucosal effects of EAEC, we studied strain 042, which we have shown to cause diarrhea in adult volunteers. Strain 042 was incubated in an in vitro organ culture model with biopsy-derived normal intestinal mucosa from pediatric patients. Strain 042 adhered strongly to samples of jejunal, ileal, and colonic mucosa. In addition, scanning electron microscopic examination of in vitro-infected intestinal biopsies revealed cytotoxic effects marked by exfoliation of mucosal epithelial cells. To develop an in vitro model to study these effects, we incubated 042 with polarized monolayers of the human intestinal epithelial cell lines Caco-2 and T84. Strain 042 adhered strongly to T84 cells but not to Caco-2 cells. T84 cells infected with 042 displayed marked toxic effects, most prominently in areas where bacteria were adhering. The apical membrane of damaged cells exhibited vesiculation and shedding of microvilli. The cytoplasm of affected cells displayed subnuclear vacuolization, and in some cases, nuclei of affected cells became separated from the surrounding cytoplasm. Severely affected cells ruptured, releasing their nuclei. Vacuolated remnant cells were seen throughout the monolayer. Strain 042 was not internalized by T84 cells. We concluded that EAEC strain 042 alters intestinal cell morphology, ultimately leading to cell death. Although the factor(s) required for this effect remains to be elucidated, T84 cells may serve as a valuable model in EAEC pathogenesis studies.

EAggEC are an important cause of persistent diarrhoea in the developing world, however, pathogenic mechanisms remain unclear. Animal models have suggested that EAggEC may damage the intestinal mucosa and putative toxins have been described. We have used in vitro organ culture of histologically normal human intestinal mucosa with EAggEC to assess their cytotoxic potential. Strains examined included 1) a prototype and 4 wild type EAggEC isolates from children with diarrhoea in Delhi, India, 2) two prototype and 5 wild type EAggEC strains from cases of diarrhoea in London, UK, 3) strain O42 (O44:H18), which expresses AAF/II fimbriae and causes diarrhoea in adult volunteers, in three forms — a) genetically intact, b) a TnphoA mutant which lacked AAF/II fimbrial expression, and c) an adherence-plasmid cured derivative. Group 1 strains were incubated with adult colonic mucosa, group 2 and 3 strains were incubated with small and large paediatric intestinal mucosal samples. Tissue was studied by electron microscopy following incubation. Group 1: four strains adhered to the mucosal surface and showed non-intimate attachment characteristic of fimbrially-mediated adhesion; beneath sites of adhesion vesiculation and loss of microvillus membrane were apparent. Group 2: all strains adhered to paediatric intestine — 5 bound to jejunum without morphological effects; 7 bound to ileum and induced rounding and extrusion of epithelial cells without evidence of microvillus changes; 5 adhered to colonic mucosa producing rounding-up and extrusion of epithelial cells, in addition 3 caused microvillus vesiculation, dilatation of crypt openings and increased mucus discharge from goblet cells. Group 3: strain 3a adhered to both small and large intestinal mucosa and produced similar overt changes to colonic mucosa as the latter 3 strains in group 2; strain 3b, although it did not show adherence, produced similar, but milder, mucosal changes; strain 3c did not adhere and mucosal changes were not apparent. In conclusion, EAggEC strains have a cytotoxic effect on human intestinal mucosa in vitro. This may be the basis of EAggEC induced mucosal damage.

Organ cultures of small- and large-intestinal mucosa from children were used to examine the interactions of enteroaggregative Escherichia coli (EAEC) with human intestine. Mucosae from patients aged between 3 and 190 months were cultured with five EAEC strains isolated from infants with diarrhea in the United Kingdom and with two well-described prototype EAEC strains, 17-2 and 221. The prototype strains adhered to jejunal, ileal, and colonic mucosae. The wild-type strains also adhered to this tissue but showed a variable pattern of adhesion: two adhered to all intestinal levels, one adhered to jejunum and ileum, one adhered to ileum only, and one adhered to ileum and colon. Adherence was in an aggregative or stacked-brick pattern, resembling that seen on HEp-2 cells. Electron microscopy of infected small intestinal mucosa revealed bacteria in association with a thick mucus layer above an intact enterocyte brush border, which contained extruded cell fragments. This mucus layer was not present on controls. EAEC adherence to colonic mucosa was associated with cytotoxic effects including microvillous vesiculation (but without evidence of an attaching/effacing lesion), enlarged crypt openings, the presence of intercrypt crevices, and increased epithelial cell extrusion. These results demonstrate that in vitro organ culture of intestinal mucosa from children can be used to investigate EAEC pathogenesis in childhood directly. EAEC strains appear able to colonize many regions of the gastrointestinal tract, without overt changes to small intestinal mucosa but with cytotoxic effects on colonic mucosa.

The formation of attaching and effacing (AE) lesions is central to the pathogenesis of enteropathogenic Escherichia coli (EPEC)-mediated disease in humans and Citrobacter rodentium-mediated transmissible colonic hyperplasia in mice. Closely related outer membrane proteins, known as intimins, are required for formation of the AE lesion by both epec and C. rodentium. In this study we found similar ultrastructural damage in small intestinal biopsies from an EPEC-infected child and large bowel specimens from C. rodentium-infected mice. The C. rodentium-infected large bowel biopsies revealed massive hyperplastic reactions and the infected human small intestinal biopsies showed an increase in total crypt cell number and mitotic index. EPEC-infected small intestinal organ cultures revealed bacteria adhering in a localized pattern and evidence of AE lesions. Covaspheres® coated with a biologically active cell-binding domain of intimin also adhered to cells in a localized fashion but did not induce the characteristic AE lesions.

Journal of Pediatric Gastroenterology and NutritionVolume 20, Issue 4 p. 461-461 Abstract: PDF Only 66 ENTEROPATHOGENIC AND ENTEROAGGREGATIVE E COLI IN VIVO AND IN VITRO S Hicks, S Hicks Academic Department of Paediatric Gastroenterology, Queen Elizabeth Hospital for Children, London, UKSearch for more papers by this authorS Savarino, S Savarino Enteric Diseases Program, Naval Medical Research Institute, Rockville, USASearch for more papers by this authorJ Walker-Smith, J Walker-Smith Academic Department of Paediatric Gastroenterology, Queen Elizabeth Hospital for Children, London, UKSearch for more papers by this authorA D Phillips, A D Phillips Academic Department of Paediatric Gastroenterology, Queen Elizabeth Hospital for Children, London, UKSearch for more papers by this author S Hicks, S Hicks Academic Department of Paediatric Gastroenterology, Queen Elizabeth Hospital for Children, London, UKSearch for more papers by this authorS Savarino, S Savarino Enteric Diseases Program, Naval Medical Research Institute, Rockville, USASearch for more papers by this authorJ Walker-Smith, J Walker-Smith Academic Department of Paediatric Gastroenterology, Queen Elizabeth Hospital for Children, London, UKSearch for more papers by this authorA D Phillips, A D Phillips Academic Department of Paediatric Gastroenterology, Queen Elizabeth Hospital for Children, London, UKSearch for more papers by this author First published: 01 May 1995 https://doi.org/10.1002/j.1536-4801.1995.tb11650.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume20, Issue4May 1995Pages 461-461 RelatedInformation