Search

BACKGROUND NlmCategory: BACKGROUND content: Diarrheagenic Escherichia coli (DEC) are among the leading pathogens associated with endemic diarrhea in low income countries. Yet, few epidemiological studies have focused the contribution of enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC). - Label: METHODS NlmCategory: METHODS content: "We assessed the contribution of EHEC, EIEC and DAEC isolated from stool samples from a case-control study conducted in children aged

Background The relationship of diffusely adherent Escherichia coli (DAEC) with pediatric inflammatory bowel disease (IBD) has not been previously studied. Diffusely adherent E. coli are a common cause of long-lasting childhood diarrhea and we postulated that they may induce inflammation of the intestinal mucosa, contributing to the development of IBD in susceptible children. Objectives The aim of the study was to investigate the relationship between DAEC and pediatric IBD, including Crohn's disease (CD) and ulcerative colitis (UC). Diffusely adherent E. coli isolates were also assessed regarding their pathogenicity. Material and methods Diffusely adherent E. coli were screened among 130 E. coli strains isolated from intestinal biopsy specimens from 26 children with IBD using polymerase chain reaction (PCR) with primers specific to the pathotype and adherence assays to HEp-2 cells. Diffusely adherent E. coli were further analyzed for their ability to adhere to and invade polarized Caco-2 cells. The immunomodulatory effect of DAEC on the secretion of tumor necrosis factor α (TNF-α) by human monocyte-derived macrophages (MDM) was assessed using an immunoenzymatic assay. Results Diffusely adherent E. coli were recovered from 18 (69.2%) of the 26 intestinal biopsy specimens from both CD and UC patients. Most DAEC isolates carried AfaE3 adhesin, adhered to and were internalized by Caco-2 cells, and induced secretion of elevated levels of TNF-α. Conclusions The study demonstrated the internalization of DAEC by intestinal epithelial cells and their ability to induce secretion of increased level of TNF-α in a Caco-2/macrophage compartmentalized culture. This indicated that the pathovar should be considered a pathobiont inducing inflammation of the intestinal mucosa in pediatric patients with IBD.

The aim of the study was to determine the frequency of six genes encoding serine protease autotransporter proteins Enterobacteriaceae (SPATE) in diffusely adherent Escherichia coli (DAEC) isolates from children with (WD, n=63) and without diarrhea (WOD, n=41) from Lima, Peru. WOD were considered a control group. For the detection of the genes, 2 multiple PCRs were standardized: triple A (sigA, pet, espP) and triple B (sat, pic, espC). In both groups, the most frequent SPATE gene was Sat (39.7% of WD and 41.5% of WOD), followed by spP (20.6% and 9.7% in WD and WOD respectively). The other genes were detected in proportions lower than 10.0%, in the following order of frequency: pet, sigA, espC and pic, without significant differences between the groups. It was concluded that Sat is the most frequent SPATE in DAEC and that these strains may possess SPATE genes regardless of whether they are isolated in WD or WOD. El objetivo del estudio fue determinar la frecuencia de seis genes que codifican proteínas autotransportadoras serin-proteasa de Enterobacteriaceae (SPATE) en aislamientos de Escherichia coli difusamente adherente (DAEC) provenientes de niños con diarrea (NCD, n=63) y sin diarrea (NSD, n=41) de Lima, Perú. Los NSD se consideraron como grupo control. Para la detección de los genes se estandarizaron 2 PCRs múltiples: triple A (sigA, pet, espP) y triple B (sat, pic, espC). En ambos grupos el gen SPATE más frecuente fue sat (39,7% de NCD y 41,5% de NSD), seguido de espP (20,6% y 9,7% en NCD y NSD respectivamente). Los otros genes se detectaron en proporciones inferiores al 10,0%, en el siguiente orden de frecuencia: pet, sigA, espC y pic, sin diferencias significativas entre los grupos. Se concluye que Sat es la SPATE más frecuente en cepas DAEC, y que estas cepas pueden poseer genes SPATE independientemente de si se aíslan en NCD o NSD

Diarrhoea is a major health concern, especially in developing countries. Research has implicated diffusely adherent Escherichia coli (DAEC) strains as a cause of diarrhoea. In this study, we investigated the prevalence, adherence assay, virulence gene profiles and antimicrobial resistance of DAEC at a hospital in southern Iran. In this cross-sectional study, 309 infants and children under the age of 13 years with diarrhoea who had been referred to Shahid Dastgheib Hospital, Shiraz between October 2018 and May 2019 were recruited. Microbiological methods, PCR, HEp-2 adherence assay and antimicrobial susceptibility test were used. Of the 309 stool samples, 207 (66.9%) were found to contain E. coli by biochemical tests and culture. Molecular analysis of Afa/Dr and AIDA-I adhesin-encoding genes showed that 14 (6.7%) out of 207 E. coli isolates were DAEC. All DAEC isolates in HEp-2 cells showed a diffusely adherent pattern. The virulence genes sat, pet, sigA, pic, astA and fimH were found in 50%, 0%, 14.2%, 14.2%, 21.4% and 100% of DAEC isolates, respectively. The most effective antibiotic against the DAEC isolates was imipenem (92.8%) and the least effective was ampicillin (0%). Our findings expand the knowledge on DAEC prevalence and its characteristics in Iran. It also explains the role of virulence genes in DAEC pathogenesis. The results showed that although the prevalence of DAEC is low, these strains exhibit a high rate of antimicrobial resistance as well as high frequency for carrying virulence genes.

Diarrheagenicity of diffusely adherent Escherichia coli (DAEC) remains controversial. Previously, we found that motile DAEC strains isolated from diarrheal patients induced high levels of interleukin 8 (IL-8) secretion via Toll-like receptor 5 (TLR5). However, DAEC strains from healthy carriers hardly induced IL-8 secretion, irrespective of their possessing flagella. In this study, we demonstrated that SK1144, a DAEC strain from a healthy carrier, suppressed IL-8 and IL-6 secretion from human epithelial cell lines. Suppression of IL-8 in human embryonic kidney (HEK293) cells that were transformed to express TLR5 was observed not only upon inflammatory stimulation by flagellin but also in response to tumor necrosis factor alpha (TNF-α) and phorbol myristate acetate (PMA), despite the fact that the TNF-α- and PMA-induced inflammatory pathways reportedly are not TLR5 mediated. SK1144 neither decreased IL-8 transcript accumulation nor increased intracellular retention of IL-8. No suppression was observed when the bacteria were cultured in Transwell cups above the epithelial cells; however, a nonadherent bacterial mutant (lacking the afimbrial adhesin gene) still inhibited IL-8 secretion. Direct contact between the bacteria and epithelial cells was necessary, but diffuse adhesion was dispensable for the inhibitory effects. Infection in the presence of chloramphenicol did not suppress cytokine release by the epithelial cells, suggesting that suppression depended on effectors synthesized de novo Inflammatory suppression was attenuated with infection by a bacterial mutant deleted for hcp (encoding a component of a type VI secretion system). In conclusion, DAEC strains from healthy carriers impede epithelial cell cytokine secretion, possibly by interfering with translation via the type VI secretion system.

We report here Illumina-corrected PacBio whole-genome sequences of an Escherichia coli serotype O157:H7 strain (2017C-4109), an E. coli serotype O[undetermined]:H2 strain (2017C-4173W12), and a Salmonella enterica subsp. enterica serovar Enteritidis strain (2017K-0021), all of which carried the mcr-1 resistance gene on an IncI2 or IncX4 plasmid.

A total of 220 enteroadherent Escherichia coli were identified from 729 Egyptian children with diarrhea using the HEp-2 adherence assay. Enteropathogenic E.coli (EPEC = 38) was common among children

Diffusely adherent Escherichia coli (DAEC) is thought to cause diarrhoea in children, and so too are other diarrhoeagenic E. coli (DEC); however, the evidence base is inconclusive. DEC pathotypes are differentiated on the basis of their pathogenic features, and thus cannot be quickly identified on selective culture media. Molecular techniques, not readily available in most clinical laboratories, are required to differentiate DEC strains from non-pathogenic E. coli in the stool flora. We report a case of persistent bloody diarrhoea, without fever, in a previously healthy 21-month infant from whom we isolated five DAEC strains. The child’s stools movements were loose, with gross blood and mucus; fresh mount analysis revealed numerous faecal leukocytes and erythrocytes. Response to antimicrobial treatment with trimethoprim-sulfamethoxazole was poor despite susceptibility in vitro. Although the patient improved with azithromycin, blood was present in the patient’s stools for over 30 days. The severe diarrhoea in this patient might be explained by the fact that these DAEC isolates harboured a siderophore receptor, which allows the bacteria to use iron derived from haem compounds that promote its multiplication. The isolates also induced in vitro secretion of several immunomodulatory cytokines that may account for the patient’s loose stools and faecal leukocytes. DAEC may play a greater role than suspected in afebrile children with bloody diarrhoea.

Background: Diarrhea continues to be one of the most common causes of morbidity and mortality among children in developing countries. Recently, some studies have implicated diffusely adherent Escherichia coli (DAEC) strains as a cause of diarrhea. The clinical manifestations of diarrhea caused by DAEC strains may include watery or bloody diarrhea, abdominal pain, dehydration, and fever. Objectives: The aims of this study were (1) isolation of E. coli from fecal samples obtained from patients with diarrhea in Shiraz (Iran), (2) detection of DAEC pathotypes in isolates by molecular diagnostic techniques such as conventional PCR and real-time PCR assays, and (3) investigation of the antibiotic susceptibility of DAEC isolates. Materials and Methods: Seven hundred and fifteen stool specimens of diarrhea patients were collected in Shiraz, Fars, Iran. Diarrheagenic E. coli strains were isolated by standard biochemical analysis. Susceptibility testing was performed by the diffusion method, according to the guidelines of the Clinical and Laboratory Standards Institute. Real-time PCR and conventional PCR were used to detect the daaD gene in the DAEC strains isolated. Results: Of the 715 stool samples tested, 101 (14.1%) were identified as E. coli by biochemical tests and culture. Of the infected patients, 58 (57%) were male and 43 (43%) were female, with a mean age of 13.52 (SD, 1.66) years. Eight of the E. coli strains were identified as DAEC strains. The most effective antibiotics against the DAEC isolated were levofloxacin and imipenem, and the least effective antibiotics were ampicillin and penicillin. Conclusions: Our analysis indicated that DAEC strains may be considered as potential pathogens in Shiraz, southern Iran. Further, although the prevalence of DAEC is low, prevention of infection caused by this bacterium among asymptomatic patients is crucial. Therefore, further characterization of the different virulence aspects of DAEC strains is required.

DAEC is considered potentially diarrheagenic. For diffuse adhesion, the role of the Afa, which was originally identified as a uropathogenic factor, is now understood. However, the role of DAEC in diarrheal disease remains controversial because DAEC is often isolated not only from patients but also from healthy individuals. Previously, we suggested that Afa/Dr DAEC, which can induce high levels of IL-8 secretion in cultures of human carcinoma epithelial cells (HEp-2, Caco-2), is enterovirulent. In the present study, we examined whether IL-8 secretion induced by certain Afa/Dr DAEC strains was primarily due to flagella via TLR5. All IL-8 high-inducing strains were highly motile in swarming tests. Partially purified flagella induced IL-8 in a dose-dependent manner. However, IL-8 induction was inhibited by small-interfering RNA against TLR5 or by treating flagella with disialoganglioside-GD1a, a TLR5 blocker. TLR5 is reportedly located on the basolateral side of intestinal epithelia; flagella should not have reached TLR5 from the apical side beyond tight junctions. Reduction in the number of intracellular organisms by wortmannin, a PI3K inhibitor, did not reduce IL-8 secretion. Afa/Dr DAEC seemed to loosen the tight junctions because it quickly reduced transepithelial electrical resistance after infection. Decreased resistance led to increased IL-8 production. In conclusion, diffuse adhesion itself is insufficient to induce high levels of IL-8, and simultaneous stimulation by flagella via TLR5 is likely required for additional induction. Clinically, high motility may be a candidate criterion for predicting the ability of Afa/Dr DAEC strains to induce higher levels of IL-8 secretion.

Seven hundred and thirty-five diarrhoeic faecal samples from children were investigated for presence of enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), diffusely adherent E. coli (DAEC) and Salmonella spp. by polymerase chain reaction (PCR) and bacterial culture. Out of 675 samples from Kashmir, 55 isolates were obtained, which carried at least one virulence gene studied. Out of the 55 isolates, 36 (65.45%) were EAEC, 18 (32.72%) were ETEC while only one isolate (1.81%) was DAEC. All the EAEC isolates were found to be typical as they possessed aggR gene. Six (16.66%) EAEC isolates carried the astA gene. Out of the 18 ETEC isolates, 13 carried the elt gene alone, four possessed both the elt and est genes and the remaining one harboured the est gene alone. Five ETEC isolates also possessed astA gene. Nineteen EAEC isolates belonged to 10 different serogroups. Serogroup O153 was most frequent. The ETEC isolates also belonged to 10 different serogroups of which O159 was most predominant. Out of 224 E. coli isolates from 60 samples of Secunderabad, 27 isolates carried at least one virulence gene. Out of 27 isolates 22 (81.48%) were typical EAEC, three (11.11%) were ETEC and two (7.4%) were DAEC. Fifteen EAEC isolates belonged to seven different serogroups with O86 as most frequent. Four EAEC isolates also possessed the astA gene. All the three ETEC isolates harboured elt gene only and belonged to three different serogroups. Two isolates of Salmonella Worthington were obtained from only two samples in Kashmir.

Diarrhea is a leading cause of illness and death, particularly in developing countries. Enteropathogenic Escherichia coli, enterotoxigenic E. coli (ETEC), enteroinvasive E. coli, enterohemorrhaghic E. coli (EHEC), enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC) have been associated with diarrheal disease in different parts of Africa, particularly among young children, HIV-positive individuals, and visitors from abroad. Each of these E. coli pathotypes uses distinct mechanisms, which are only partially understood, to colonize infected hosts and produce diarrhea. All known diarrheagenic E. coli pathotypes have been reported from diverse locations within Africa but the true burden from these pathogens is unknown because very few studies seeking these organisms with discriminatory methodology have been performed. Recent reports implicate ETEC and EAEC in a considerable proportion of childhood and travellers' diarrheas and suggest that some sub-types of these categories may have greater epidemiological significance than others. The significant contribution of EHEC to bloody diarrhea and hemolytic uremic syndrome is underappreciated because diagnostic capacity for this pathotype is generally inferior to that for confounders such as Shigella and Entamoeba. Recent studies in Africa have revealed the worrisome emergence of antimicrobial resistance and high asymptomatic carriage rates for diarrheagenic E. coli but bacterial and host factors that predispose to disease, as well as non-human reservoirs, are largely unknown. Future diarrheal disease research needs to focus on broadening the repertoire of pathogens sought in epidemiological surveys to include multiple categories of diarrheagenic E. coli while building capacity to detect these pathogens in local reference laboratories.

The innate immune response to enteropathogenic bacteria includes chemokine-induced polymorphonuclear neutrophil (PMN) migration across mucosal epithelia leading to bacterial clearance and resolution of infection. Among these bacteria, diffusely adherent Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC), causing childhood diarrhea, can promote IL-8-dependent PMN transmigration across cultured intestinal epithelial cell monolayers via MAPK pathway activation. However, interactions between PMN and Afa/Dr DAEC are poorly documented and constitute the aim of the present study. Using the human PLB-985 cell line differentiated into fully mature PMN, we described the coordinated response to various E. coli. The rapid and strong release of reactive oxygen species and preformed intragranular mediators (myeloperoxidase and IL-8) is followed by a later TNF-α, IL-1β, and IL-8 synthesis. The use of wild-type (IH11128, C1845, LF82), control (AAEC185), and recombinant (AAEC185 bearing Dr or F1845 fimbriae, AdLF82, or type 1 pili) bacterial strains allowed us to demonstrate that late IL-8 hyperproduction is triggered by type 1 pili but not by Dr or F1845 fimbriae; MAPKs (p38, ERK, Src) and NF-κB activations are implicated in this response. Thus, in the course of Afa/Dr DAEC intestinal infection, epithelium- and neutrophil-derived IL-8 could, at least in part, control the flow of neutrophils through the lamina propria. Afa/Dr DAEC-induced IL-8 hyperproduction by PMN might thus be important for inducing and perpetuating local inflammation, and this self-amplifying loop might play a role in the pathogenesis of inflammatory bowel diseases such as Crohn’s disease.

Background Enteropathogens cannot be identified in 40% to 50% of subjects with travelers’ diarrhea (TD). Methods We used polymerase chain reaction (PCR) methods to look for the presence of two bacterial causes of diarrhea in a large group of international travelers after failing to detect a pathogen by conventional tests. DNA was isolated from the diarrheal stool and subjected to PCR from 162 subjects from whom we earlier failed to identify a pathogen in a previous study and included 54 from Antigua, Guatemala, 39 from Guadalajara, Mexico, 29 from Kolkata, India, and 40 from Goa, India. Gene products for enterotoxigenic Escherichia coli (ETEC)—LT (heat‐labile enterotoxin) and ST (heat‐stable enterotoxin)—and diffusely adherent E coli (DAEC), afa/dr (Afa fimbrial and Dr nonfimbrial family of adhesins), were used. Results At least one gene product was identified in diarrhea stool samples of 47 of 162 (29%) subjects. ETEC virulence genes (LT, ST) were found in 34 (21%) samples studied, with rates of occurrence ranging from 8% in Goa to 39% for the samples from Guatemala ( p = 0.0006). A large number of ST‐only strains explained the high ETEC rate in Guatemala. DAEC afa / dr family of adhesions was identified in between 8 and 14% of the samples. Conclusions ETEC and DAEC were implicated in nearly one‐third of the subjects initially diagnosed as pathogen negative. Direct PCR results from stools are consistent with the previous assumption that most undiagnosed TD is bacterial in nature and also highlights the potential value that PCR can add to studies designed to evaluate treatment and preventive interventions for TD, including vaccines.

Diffusely adherent Escherichia coli (DAEC) were the last diarrheagenic E. coli pathogroup (DEC) to be recognized. DAEC pathogroup encompasses a heterogeneous group of E. coli strains, harboring genes encoding for Afa/Dr adhesins that are capable of causing diarrhea illness in otherwise healthy individuals. Afa/Dr family includes Afa, Dr, and F1845 adhesins that are encoded by the afa/dra/daa operons, respectively. Afa/Dr adhesins bind to cell receptors human decay-accelerating factor (hDAF) and carcinoembryonic antigen cell adhesion molecules (hCEACAMs), inducing receptor clustering and finger-like cell projections, resulting in bacteria embedding without complete internalization. DAEC infection also causes damages on epithelial cells, including loss of microvilli structure, impaired enzyme activities of functional brush border-associated proteins, and loss of adherens junctions, in an Afa/Dr-dependent fashion. Diarrhea prevalence studies among Latin American children have confirmed that DAEC diarrhea illness susceptibility is age-related and DAEC-diarrhea risk increases with children’s age. DAEC was the most prevalent DEC identified from children with acute diarrhea attending a hospital rehydration unit and an Emergency Room service in Mexico and US, respectively. In adults, DAEC was identified in travelers’ diarrhea patients who visited Latin America and from HIV-positive patients with diarrhea from Peru. Recently, DAEC strains carrying virulence genes associated with pathogenesis, M cell translocation, angiogenesis, and genotoxicity were isolated from patients with inflammatory bowel disease and colorectal cancer. DAEC is a bacterial pathogen that induces unique alterations on epithelial cells, resulting in diarrhea illness and epithelia damage that may also contribute to the development of other intestinal diseases.

The role of diffusely adherent Escherichia coli (DAEC) in diarrheal disease has been controversial. However, DAEC strains were recently implicated in diarrheal disease in developing countries. To clarify whether DAEC are prevalent among sporadic cases of diarrheal illness in Osaka City, Japan, E. coli strains isolated between July 1997 and March 2000 during diarrheagenic E. coli (DEC) investigation were retrospectively examined. DAEC strains were recognized among 41 (4.4%) of 924 patients and formed the biggest subgroup of DEC. Previously, we reported that some DAEC strains caused epithelial cells to secrete as much IL-8 as enteroaggregative E. coli strains did. In this study, we attempted to evaluate epidemiologically whether the ability of DAEC to induce IL-8 was involved in the pathogenesis. Relationship among patient age, symptoms, Afa adhesins, season and IL-8 induction were examined. The subgroup of DAEC that possessed Afa genes and/or induced a high level of IL-8 was significantly prevalent among patients age 1 to 4 years; however total DAEC was not significantly high among the children compared to other age group. IL-8 inducing DAEC seems to play a role in causing sporadic diarrheal illnesses, particularly in pediatric fields. Investigations highlighting the relationship between IL-8 induction and enteropathogenicity are clearly necessary to confirm the role of DAEC in infectious enteritis.

Escherichia coli that adhere sparsely to human epithelial (HEp-2) cells are known as diffusely adherent E. coli (DAEC) and considered potentially diarrheagenic. The role of the afimbrial adhesive sheath (Afa)—identified originally as a uropathogenic factor—in diffuse adhesion is now understood. However, the role of DAEC in diarrheal disease remains controversial. Recently, ability to induce interleukin-8 (IL-8) secretion from intestinal epithelial cells has been suggested as one of the properties of enterovirulent bacteria. In this study, we examined whether DAEC strains possessing Afa genes induced IL-8 in cultures of human carcinoma epithelial cells (e.g., HEp-2, Caco-2, and T84). Nineteen afa-positive DAEC strains were examined for their ability to induce IL-8 secretion, and only 7 strains (37%; 7/19) induced IL-8 as much as enteroaggregative E. coli did. No marked differences in adhesion were observed between high and low inducers. Diffusive adhesiveness itself is unlikely to be sufficient to induce IL-8. All high inducers were motile and others were nonmotile. Additional stimulation by flagella may be required to cause high levels of chemokine induction. Motility or presence of flagella can be an important criterion to predict DAEC diarrheagenicity at clinical laboratories.

SUMMARYOver the last few years, dramatic increases in our knowledge about diffusely adheringEscherichia coli(DAEC) pathogenesis have taken place. The typical class of DAEC includesE. colistrains harboring AfaE-I, AfaE-II, AfaE-III, AfaE-V, Dr, Dr-II, F1845, and NFA-I adhesins (Afa/Dr DAEC); these strains (i) have an identical genetic organization and (ii) allow binding to human decay-accelerating factor (DAF) (Afa/DrDAFsubclass) or carcinoembryonic antigen (CEA) (Afa/DrCEAsubclass). The atypical class of DAEC includes two subclasses of strains; the atypical subclass 1 includesE. colistrains that express AfaE-VII, AfaE-VIII, AAF-I, AAF-II, and AAF-III adhesins, which (i) have an identical genetic organization and (ii) do not bind to human DAF, and the atypical subclass 2 includesE. colistrains that harbor Afa/Dr adhesins or others adhesins promoting diffuse adhesion, together with pathogenicity islands such as the LEE pathogenicity island (DA-EPEC). In this review, the focus is on Afa/Dr DAEC strains that have been found to be associated with urinary tract infections and with enteric infection. The review aims to provide a broad overview and update of the virulence aspects of these intriguing pathogens. Epidemiological studies, diagnostic techniques, characteristic molecular features of Afa/Dr operons, and the respective role of Afa/Dr adhesins and invasins in pathogenesis are described. Following the recognition of membrane-bound receptors, including type IV collagen, DAF, CEACAM1, CEA, and CEACAM6, by Afa/Dr adhesins, activation of signal transduction pathways leads to structural and functional injuries at brush border and junctional domains and to proinflammatory responses in polarized intestinal cells. In addition, uropathogenic Afa/Dr DAEC strains, following recognition of β1integrin as a receptor, enter epithelial cells by a zipper-like, raft- and microtubule-dependent mechanism. Finally, the presence of other, unknown virulence factors and the way that an Afa/Dr DAEC strain emerges from the human intestinal microbiota as a “silent pathogen” are discussed.

Afa/Dr diffusely adheringEscherichia coli(Afa/Dr DAEC) strains cause symptomatic urinary tract and intestinal infections. The proinflammatory effects of Afa/Dr DAEC strains in vitro have been not investigated to date. In the present study, we used confluent polarized monolayers of intestinal cell line T84 to evaluate the consequences of epithelial infection by Afa/Dr DAEC strains in terms of proinflammatory response. Polymorphonuclear leukocyte (PMNL) migration across the epithelial barrier was induced after incubation of the T84 monolayers with the wild-type Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin and strain IH11128 harboring the Dr hemagglutinin, and theE. colilaboratory strain HB101 was transformed with the pSSS1 plasmid, producing Afa/Dr F1845 adhesin. PMNL migrations were correlated with a basolateral secretion of interleukin-8 by T84 cells and were abolished after incubation of epithelial cells with an anti-decay accelerating factor (DAF) antibody that recognized the short consensus repeat 3 domain of DAF (monoclonal antibody 1H4). Moreover, Afa/Dr DAEC strains induced tyrosine phosphorylation of several T84 proteins and activated the mitogen-activated protein kinases (ERK1/2 mitogen-activated protein, P38, and Jun-C kinases). These data demonstrated for the first time that, in vitro, Afa/Dr DAEC strains exert a proinflammatory signal in intestinal epithelial cells.

MICA are distant homologs of MHC class I molecules expressed in the normal intestinal epithelium. They are ligands of the NKG2D activating receptor expressed on most γδ T cells, CD8+ αβ T cells, and natural killer cells and therefore play a critical role in innate immune responses. We investigated MICA cell-surface expression on infection of epithelial cell lines by enteric bacteria and show here that MICA expression can be markedly increased by bacteria of the diffusely adherentEscherichia colidiarrheagenic group. This effect is mediated by the specific interaction between bacterial adhesin AfaE and its cellular receptor, CD55, or decay-accelerating factor. It is extremely rapid after AfaE binding, consistent with a stress-induced signal. MICA induction on epithelial cells triggered IFN-γ release by the NKG2D expressing natural killer cell line NKL. This host–bacteria interaction pathway could play a role in the pathogenesis of inflammatory bowel disease, a condition that implicates a bacterial trigger in genetically susceptible individuals. This was supported by the increased MICA expression at the surface of epithelial cells in colonic biopsies from Crohn's disease-affected patients compared with controls.

This chapter focuses on diarrheagenic Escherichia coli strains and the various pathogenicity islands (PAIs) and other mobile genetic elements that differentiate these pathogens from normal-flora E. coli. At least six categories of diarrheagenic E. coli strains have been defined. Five of these categories, enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC), are reviewed in the chapter. A sixth category, diffusely adherent E. coli (DAEC), is a heterogeneous group of organisms, and epidemiological studies have given conflicting results about the true clinical significance of these strains. Mobile genetic elements encoding virulence factors in human diarrheagenic E. coli are listed in the chapter. The majority of EPEC strains associated with diarrhea possess the EPEC adherence factor (EAF) plasmid and are referred to as typical EPEC strains, while EPEC strains that do not possess EAF plasmids are referred to as atypical EPEC strains. The majority of genes within this region have homology to transposons and insertion elements (IS elements), although these elements are apparently incomplete and nonfunctional. The great variety of mobile genetic elements present in diarrheagenic E. coli , including plasmids, bacteriophages, transposons, and PAIs, on a K-12 backbone with so many horizontally transferred regions indicates an enormous genomic plasticity that complicates the efforts to categorize the existing subgroups into sharply delineated pathotypes and the attempts to predict what novel combination of virulence factors may emerge in the future.

Childhood diarrheal diseases remain highly endemic in northeastern Brazil. The attributable fraction of all diarrheal diseases among children less than 2 years of age due to Escherichia coli was examined in a 2-year prospective study in two large urban centers of Brazil. Between May 1997 and June 1999, fecal E. coli isolates from 237 children with diarrhea (217 acute and 20 persistent cases) and 231 children without diarrhea (controls) attending two hospitals in Northeast Brazil were tested for their pattern of adherence to HEp-2 cells and for colony hybridization with DNA probes specific for the six pathotypes of diarrheagenic E. coli. Enteroinvasive E. coli, enterotoxigenic E. coli and enterohemorrhagic E. coli were not isolated from any children. Diffusely adherent E. coli (DAEC) and enteroaggregative E. coli (EAEC) were the most frequent isolates with similar frequencies from children with or without diarrhea. Atypical EPEC (EAF-negative) strains were isolated with similiar frequency from both cases (5.5%) and controls (5.6%). Enteropathogenic E. coli (typical EPEC) strains, characterized by localized adherence pattern of adherence, hybridization with the EAF probe, and belonging to the classical O serogroups, were significantly associated with diarrhea (P = 0.03). These E. coli strains associated with diarrhea accounted for 9% of all children with diarrhea. Collectively, in Northeast Brazil, E. coli strains comprise a small proportion of severe diarrhea prevalence in children.

Operons of the afa family are expressed by pathogenic Escherichia coli strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048–5059, 1999) was used to develop a new PCR assay for detecting all the operons of the afa family with a single genetic tool. This PCR approach was validated by investigating three collections of human E. coli isolates originating from the stools of infants with diarrhea (88 strains), the urine of patients with pyelonephritis (97 strains), and the blood of cancer patients (115 strains). The results obtained with this single test and those previously obtained with several PCR assays were closely correlated. The AfaE adhesins encoded by the afa operons are variable, particularly with respect to the primary sequence encoded by the afaE gene. The receptor binding specificities have not been determined for all of these adhesins; some recognize the Dr blood group antigen (Afa/Dr + adhesins) on the human decay-accelerating factor (DAF) as a receptor, and others (Afa/Dr − adhesins) do not. Thus, the afa operons detected in this study were characterized by subtyping the afaE gene using specific PCRs. In addition, the DAF-binding capacities of as-yet-uncharacterized AfaE adhesins were tested by various cellular approaches. The afaE8 subtype (Afa/Dr − adhesin) was found to predominate in afa -positive isolates from sepsis patients (75%); it was frequent in afa -positive pyelonephritis E. coli (55.5%) and absent from diarrhea-associated strains. In contrast, Afa/Dr + strains (regardless of the afaE subtype) were associated with both diarrhea (100%) and extraintestinal infections (44 and 25% in afa -positive pyelonephritis and sepsis strains, respectively). These data suggest that there is an association between the subtype of AfaE adhesin and the physiological site of the infection caused by afa -positive strains.

Diarrhoeagenic Escherichia coli (DEC) were sought in stool specimens from 72 adults and children aged over 3 years, who presented with diarrhoea at a hospital in Accra, Ghana, and 72 matched controls. Only diffusely-adherent E. coli were significantly associated with disease in these older individuals (P=0.029). We additionally tested 53 specimens from infants among whom DEC were collectively associated with disease (P=0.012). Enteropathogenic, enterotoxigenic and enteroaggregative E. coli, the most commonly isolated pathotypes from infants with diarrhoea, were frequently recovered from healthy adults. Asymptomatic carriage of DEC by older individuals in Accra may place young children at risk for diarrhoea.

The phylogenetics of the various pathotypes of diarrheagenic Escherichia coli are not completely understood. In this study, we identified several plasmid and chromosomal genes in the pathogenic enteroaggregative E. coli (EAEC) prototype strain 042 and determined the prevalence of these loci among EAEC and diffusely adherent E. coli strains. The distribution of these genes is analyzed within an evolutionary framework provided by the characterization of allelic variation in housekeeping genes via multilocus enzyme electrophoresis. Our data reveal that EAEC strains are heterogeneous with respect to chromosomal and plasmid-borne genes but that the majority harbor a member of a conserved family of virulence plasmids. Comparison of plasmid and chromosomal relatedness of strains suggests clonality of chromosomal markers and a limited transfer model of plasmid distribution.

We have previously shown that some Escherichia coli strains isolated from children with diarrhea present the so-called 'localized and diffused adherence (LA/DA) pattern' in which both localized adherence (LA) and diffused adherence (DA) are expressed simultaneously. in the present study, we show that the LA adherence of these strains is genetically and phenotypically similar to that so far described for enteropathogenic E. coli (EPEC) as determined by DNA hybridization and electron microscopy. On the other hand, the DA is encoded by genes not homologous to the DAEC or AIDA-I DNA probes. in addition, the LA/DA strains are able to invade eukaryotic cells both in vitro and in vivo. in the rabbit ileal loop assay their invasion capacity goes beyond the enterocyte and reaches the muscularis mucosae as determined by transmission electron microscopy. These findings suggest that the LA/DA adherence pattern may be linked to a new E. coli virulence category which in the case of the strains studied may be associated to other virulence traits that enable them to more deeply invade the intestinal mucosa. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, Brazil Universidade Federal de São Paulo, Div Pediat Gastroenterol, São Paulo, Brazil Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, Brazil Universidade Federal de São Paulo, Div Pediat Gastroenterol, São Paulo, Brazil Web of Science

Diffusely adherent Escherichia coli (DAEC) are diarrheagenic E. coli whose pathogenetic mechanisms are largely unknown. DAEC have been shown to induce an unusual phenotype upon adherence to HEp-2 cells in culture characterized by the induction of long thin membrane processes extending from the cell surface. In addition, DAEC have been shown to be protected from the bactericidal effects of gentamicin when incubated with HEp-2 cells. In our studies, we found that three DAEC strains induced formation of eukaryotic cell processes and were protected from gentamicin killing after a 3 h incubation. Pre-incubation of HEp-2 cells with colchicine or cytochalasin D prior to infection with DAEC strain C1845 resulted in decreased projection formation, suggesting that the effect was dependent upon microfilament and microtubule rearrangement. When the standard gentamicin protection assay was extended for an additional 3 h incubation in the presence of gentamicin, a greater number of DAEC survived gentamicin treatment, more eukaryotic projections were seen in association with the bacteria and the bacteria were actually observed to be «embedded» within these projections. Projection formation was not observed when the bacteria were separated from the cells by a permeable membrane or when the inoculum was inactivated by ultraviolet irradiation. Transposon Tn phoA mutants of C1845 were screened for decreased gentamicin protection. All three mutants which were deficient in gentamicin protection demonstrated less projection formation. Insertion mutations affecting gentamicin protection were localized to both the chromosome (two) and a plasmid (one). Eukaryotic projections are a novel interaction of DAEC with epithelial cells, may play a role of the survival of the bacteria against host defenses and may contribute to DAEC pathogenesis. The effect is dependent upon epithelial cell contact and requires multiple bacterial genes.

Background Diffusely adherent Escherichia coli (DAEC) have been considered a diarrheagenic category of E. coli for which several potential virulence factors have been described in the last few years. Despite this, epidemiological studies involving DAEC have shown inconsistent results. In this work, two different collections of DAEC possessing Afa/Dr genes, from children and adults, were studied regarding characteristics potentially associated to virulence. Results DAEC strains were recovered in similar frequencies from diarrheic and asymptomatic children, and more frequently from adults with diarrhea (P Citrobacter freundii strain have shown an improved ability to form biofilms in relation to the monocultures. Control strains have shown a greater diversity of Afa/Dr adhesins and higher frequencies of cellulose, TTSS, biofilm formation and induction of IL-8 secretion than strains from cases of diarrhea in children. Conclusions DAEC strains possessing Afa/Dr genes isolated from children and adults represent two different bacterial populations. DAEC strains carrying genes associated to virulence can be found as part of the normal microbiota present in asymptomatic children.

In addition to the well-established and extensively studied categories of pathogenic E. coli, there are other groups of E. coli associated with diarrheal and/or inflammatory bowel diseases that have not been thoroughly characterized or which are considered newly emergent pathogens. This chapter focuses on three representatives of these groups of pathogenic E. coli and the current progress understanding their role in pathogenesis. Diffusely adherent E. coli (DAEC) are a collection of isolates that colonize the small bowel, considered potentially diarrheagenic, and that can cause childhood diarrhea. Adherent and invasive E. coli (AIEC) are a group of mucosa-associated bacteria with invasive properties in Crohn’s disease patients and can survive and replicate extensively in macrophages. Finally, E. coli O104:H4, a hybrid Shiga toxin-producing enteroaggregative E. coli, which caused a large outbreak in Europe in 2011, displays a unique combination of virulence traits resulting in the development of hemolytic uremic syndrome in an unusually large proportion of infected patients. This chapter summarizes recent progress in understanding the pathogenesis and the clinical outcome of these types of E. coli infections.

The pathogenesis of diffusely adherent Escherichia coli (DAEC) remains to be elucidated. Previously, we found that afimbrial adhesin gene (afa)-positive motile DAEC strains isolated from patients with diarrhoea induce high levels of IL-8 secretion in Caco-2 cells via toll-like receptor 5 (TLR-5), while non-motile strains did not. The aim of this study was to compare virulence properties, including the phylogenetic groups, afa subtypes, IL-8 secretion levels, and the effects on tight junctions, of DAEC strains isolated from healthy persons with those isolated from patients with diarrhoea. Induction of IL-8 secretion in Caco-2 cells was examined for a total of 36 afa-positive strains: 19 from diarrhoeal patients and 17 from healthy carriers. Irrespective of the source, all strains were classified into the phylogenetic group B2 or D, with the exception of two strains. All 7 motile strains isolated from diarrhoeal patients induced high levels of IL-8 secretion, while only 6 of 15 motile strains from healthy carriers induced IL-8 secretion to the same levels as the diarrhoeal strains. We speculated that additional virulence factors other than afa and motility cause the loosening of tight junctions that allows flagellin to reach TLR-5 located on the basolateral side of the epithelium. However, no differences in the TER and dextran permeability were observed between cells infected with diarrhoeal strains and those from healthy persons. Thus, diarrhoeagenic DAEC seems to possess additional factors, in addition to adhesin and flagellin, which can induce high IL-8 secretion.

Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli (EAEC), stIa/stIb and lt for enterotoxigenic E. coli (ETEC), eaeA for enteropathogenic E. coli (EPEC), stx1 and stx2 for Shiga toxin-producing E. coli (STEC), ipaH for enteroinvasive E. coli (EIEC), and daaD for diffusely adherent E. coli (DAEC).

We recently documented the neutrophil response to enterovirulent diffusely adherent Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC), using the human myeloid cell line PLB-985 differentiated into fully mature neutrophils. Upon activation, particularly during infections, neutrophils release neutrophil extracellular traps (NETs), composed of a nuclear DNA backbone associated with antimicrobial peptides, histones, and proteases, which entrap and kill pathogens. Here, using fluorescence microscopy and field emission scanning electron microscopy, we observed NET production by PLB-985 cells infected with the Afa/Dr wild-type (WT) E. coli strain C1845. We found that these NETs were able to capture, immobilize, and kill WT C1845 bacteria. We also developed a coculture model of human enterocyte-like Caco-2/TC7 cells and PLB-985 cells previously treated with WT C1845 and found, for the first time, that the F-actin cytoskeleton of enterocyte-like cells is damaged in the presence of bacterium-induced NETs and that this deleterious effect is prevented by inhibition of protease release. These findings provide new insights into the neutrophil response to bacterial infection via the production of bactericidal NETs and suggest that NETs may damage the intestinal epithelium, particularly in situations such as inflammatory bowel diseases.

SummaryOne hundred and eighty–one stool specimens from patients with various types of diarrhoea (135 patients) or from non-diarrhoeal controls (23 acute medical patients, 23 inflammatory bowel disease in remission) were investigated using a colony–blot DNA hybridization assay for the presence of Verocytotoxin–producing (VTEC), enteroaggregative (EAggEC) and diffusely adherent (DAEC)Escherichia coli. Twelve patients had probe–positive EAggEC in the stool and 8 of these had diarrhoea, 6 following recent travel. Eight patients had DAEC, 7 of whom had travellers diarrhoea. Six of 10 (60%) travellers with gastroenteritis, but without a recognized enteric pathogen, were positive for EAggEC (4) or DAEC (2). Five of 10 (50%) travellers with gastroenteritis related to a recognized enteric pathogen also had DAEC identified in their stool. Of the 23 acute medical control patients 11 had been abroad, 4 of these were immigrants and had EAggEC. VTEC were not found and, with one exception, immunoassays for antibodies toE. coliO 157 and O 2 lipopolysaccharides were negative.

Introduction. Diarrheagenic E. coli (DEC) are a major cause of diarrhea in children in developing countries. However, they are not part of routine diagnosis in clinical laboratories. Objectives. To determine the DEC prevalence in Peruvian children and to describe the genetic variability of these strains. Materials and methods. A total of 8 003 E. coli strains previously isolated from eight different studies of diarrhea in children, mainly from peri-urban areas of Lima, were analyzed. Diagnosis of DEC was done with Multiplex real-time PCR using genes for each of the 6 DEC groups. Conventional PCR was performed for the detection of additional virulence genes. Results. Globally, the mean prevalence in diarrhea samples (n=4,243) was: enteroaggregative E. coli (EAEC) 9.9%, enteropathogenic E. coli (EPEC) 8.5%, enterotoxigenic E. coli (ETEC) 6.9%, diffusely adherent E. coli (DAEC) 4.8%, Shiga toxin-producing E. coli (STEC) 0.8% and enteroinvasive E. coli (EIEC) 0.6%. The relative frequency of each pathogen varies according to the age and the type of study. The main pathotypes in control samples (n=3,760) were EPEC (10.9%) and EAEC (10.4%). An important variability in the virulence genes frequency and molecular resistance mechanisms for each pathotype was found, without differences between diarrhea and control groups. Conclusions. DEC are a major cause of diarrhea in Peruvian children. These pathogens are highly heterogeneous. Additional studies are required to determine the prevalence in rural areas of Peru and in severe diarrhea cases. Introducción. Las E. coli diarrogénicas (DEC) son una de las principales causas de diarrea en niños en países en vías de desarrollo. Sin embargo, no son rutinariamente diagnosticadas en los laboratorios clínicos. Objetivos. Determinar la prevalencia de las DEC en niños peruanos y describir la variabilidad genética de estas cepas. Materiales y métodos. Se utilizaron 8 003 cepas de E. coli previamente aisladas de ocho estudios previos de diarrea en niños, mayormente en zonas periurbanas de Lima. El diagnóstico de las DEC fue a través de un PCR múltiple a tiempo real para los seis grupos de DEC. Se empleó PCR para la determinación de genes adicionales de virulencia. Resultados. La prevalencia promedio global en muestras de diarrea (n=4 243) fue: E. coli enteroagregativa (EAEC) 9,9%, enteropatogénica (EPEC) 8,5%, enterotoxigénica (ETEC) 6,9%, difusamente adherente (DAEC) 4,8%, productora de toxina shiga (STEC) 0,8% y enteroinvasiva (EIEC) 0,6%. La frecuencia relativa de cada patógeno varía según la edad y tipo de estudio. Los principales patotipos en muestras control (n=3 760) fueron EPEC (10,9%) y EAEC (10,4%). Se encontró una gran variabilidad en la frecuencia de genes de virulencia para cada patotipo, así como en los mecanismos moleculares de resistencia, sin diferencias significativas entre muestras de diarrea y control. Conclusiones. Las DEC son causa importante de diarrea en niños peruanos. Estos patógenos son altamente heterogéneos. Se requieren estudios adicionales para determinar la prevalencia en zonas rurales del Perú, así como en casos graves de diarrea.

Introduction. Diffusely adherent E. coli (DAEC) is the sixth recognized group of diarrheagenic E. coli. However, its association with diarrhea remains controversial. Variability in the adherence patterns of clinical strains is unknown. Objectives. To compare the adherence patterns between strains isolated from children with and without diarrhea. Materials and methods. A total of 31 DAEC strains were analyzed, 25 from children with diarrhea and 6 from asymptomatic (control) children, isolated from a cohort study of children under one year of age in the southern districts of Lima. DAEC were identifed by PCR (daaD gene). The pattern and adherence score in HEp-2 cell culture were evaluated, Actin polimerization was determined by fuorescence actin staining (FAS) and motility was evaluated by conventional microbiology methods. Results. Diffuse adherence pattern was found in 88% of diarrhea samples and in the total of control strains. The number of bacteria adhered per cell was signifcantly lower in diarrhea samples (p

Two hundred sixty-two strains of Escherichia coli isolated from diarrheal stool specimens from infants, children, and adults hospitalized in Clermont-Ferrand, France, were studied to classify them in the previously described pathogenic groups of E. coli involved in diarrheal diseases. A total of 1.5% of them belonged to the enterotoxigenic E. coli pathotype, but none belonged to the enteroinvasive E. coli, enterohemorrhagic E. coli, or enteropathogenic E. coli pathotypes. Seventeen strains (6.5%) exhibited an aggregative pattern of adhesion to HEp-2 cells (EAggEC pathotype), but of these, three (17.6%) did not hybridize with the EAggEC DNA probe. Most of the strains involved in diarrhea belonged to the diffusely adhering E. coli group; 100 strains (38.2%) exhibited a diffuse adhesion (DA) to HEp-2 cells. Only eight strains (8.9%) from controls diffusely adhered to HEp-2 cells. The highly significant difference (P < 0.0001) between DA strains from patients and from controls suggests that the diffusely adhering E. coli strains should be considered pathogens. Only 33 of them (33%) hybridized with the previously described DA DNA probe, and only 2 (2%) hybridized with the AIDA DNA probe. Four different major proteins were observed in the bacterial surface extracts of the 33 strains positive with the DA DNA probe. In addition, 16 strains that diffusely adhered to HEp-2 cells induced a cytotoxic effect on HEp-2 cells that was characterized by pyknosis and lysis of the cytoplasmic membrane. This cytotoxic effect was correlated with the synthesis of a hemolysin. The genes involved in diffuse adhesion to HEp-2 cells were located on conjugative R plasmids in strains that did not hybridize with the DA or AIDA DNA probes.

Escherichia coli from 138 fecal samples from aboriginal children, in whom no other enteric pathogen was isolated (including enterovirulent E. coli), were examined for HEp-2 cell adhesion. Twenty-five (36.8%) of 68 children with diarrhea and 32 (45.7%) of 70 without diarrhea had diffusely adherent isolates, which were thus not associated with diarrhea (P.25). However, after age stratification, childrenor = 18 months showed a significant association of diffusely adherent E. coli with diarrhea (Por = .05). Enteroaggregative E. coli were isolated from 12 children with diarrhea (17.6%) and 15 without diarrhea (21.4%); thus, there was no association with diarrhea (Por = .5). Sixteen children with diarrhea (23.5%) and 6 without diarrhea (8.6%) carried isolates that caused detachment of the HEp-2 cell monolayer from the glass coverslip when examined in the adhesion assay and were significantly associated with diarrhea (Por = .05). These isolates, termed cell-detaching E. coli, were different from all recognized classes of enterovirulent E. coli.

The prevalence of diarrheagenic Escherichia coli (DEC) in children under 5 years was studied in children with diarrhea and controls in South India. Four polymerase chain reaction (PCR) "schemes" were used to detect genes of the 6 pathotypes of DEC. In 394 children with diarrhea, 203 (52%) DEC infections were found. Among the 198 controls, 126 (63%) DEC infections were found. Enteroaggregative E. coli was the most common pathotype by multiplex PCR both in cases (58, 14.7%) and controls (47, 23.7%), followed by enteropathogenic E. coli seen in 10% cases and 8% of controls. Enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) were found in 4.1%, 2.0%, 1.0%, and 0.5% of cases, respectively. ETEC was found in 2.5% of controls, but EHEC, EIEC, and DAEC were not detected. Overall, no single assay worked well, but by discounting genes with a pathogenicity index of less than 1, it was possible to use the PCR assays to identify DEC in 75/394 (19%) cases and 12/198 (6.1%) controls, while mixed infection could be identified in 8/394 (2%) cases and 2/198 (1%) controls.

Autotransporters are an extensive family of large secreted virulence-associated proteins of gram-negative bacteria. Secretion of such large proteins poses unique challenges to bacteria. We demonstrate that autotransporters from a wide variety of rod-shaped pathogens, including IcsA and SepA of Shigella flexneri , AIDA-I of diffusely adherent Escherichia coli , and BrkA of Bordetella pertussis , are localized to the bacterial pole. The restriction of autotransporters to the pole is dependent on the presence of a complete lipopolysaccharide (LPS), consistent with known effects of LPS composition on membrane fluidity. Newly synthesized and secreted BrkA is polar even in the presence of truncated LPS, and all autotransporters examined are polar in the cytoplasm prior to secretion. Together, these findings are consistent with autotransporter secretion occurring at the poles of rod-shaped gram-negative organisms. Moreover, NalP, an autotransporter of spherically shaped Neisseria meningitidis contains the molecular information to localize to the pole of Escherichia coli . In N. meningitidis , NalP is secreted at distinct sites around the cell. These data are consistent with a model in which the secretion of large autotransporters occurs via specific conserved pathways located at the poles of rod-shaped bacteria, with profound implications for the underlying physiology of the bacterial cell and the nature of bacterial pathogen-host interactions.

Bacterial diarrheal diseases remain a major cause of morbidity and mortality in developing countries. Diffusely adhering Escherichia coli (DAEC) is a newly proposed category of diarrheagenic E. coli based on epidemiological studies. Sat, a new virulence factor of some uropathogeic Escherichia coli, was described with a vacuolating cytotoxic action in bladder and kidney tissues. In the present study, we analyzed the Sat effects, produced by a DAEC strain in rabbit ileal intestinal tissue and cultured epithelial cells. We observed enterotoxic activity in rabbit ileum tissues by Ussing chamber assays, a pronounced fluid accumulation in rabbit ileum loops with villous necrosis observed in the histopathologic examination, and morphological changes in monolayer cultures of Y1 adrenal cells. Our results suggest that DAEC strains may be involved in diarrhea.

One hundred twelve diffusely adherent Escherichia coli strains isolated from children in a case control study were evaluated for virulence-associated characteristics, serotyping, antibiotic resistance, and plasmid profiles. Half of the strains hybridized with the probes for icuA (aerobactin) and fimH (type 1 pili); daaE (F1845 fimbriae), afa (afimbrial Dr adhesin), agg-3A (aggregative adhesion fimbria type III fimbriae), pap (P fimbriae), astA (EAST1 toxin), and shET1 ( Shigella enterotoxin 1) sequences were present in shET1 gene was noted most frequently in strains isolated from patients. A minority (7%) of the strains produced hemolysin or colicin or showed cytotoxic effects on Vero cells. Forty-five different serotypes were found. The majority (70%) of the strains presented multiple antibiotic resistance. Antibiotic resistance and diffuse adherence were located on the same conjugative plasmids. These results suggest that the transfer of these potential virulence markers could be important in the epidemiology of diffusely adherent E. coli .

Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes.