Diffusely Adherent Escherichia Coli Strains Research Articles

New Insights Into DAEC and EAEC Pathogenesis and Phylogeny.

Diarrheagenic E. coli can be separated into six distinct pathotypes, with enteroaggregative (EAEC) and diffusely-adherent E. coli (DAEC) among the least characterized. To gain additional insights into these two pathotypes we performed whole genome sequencing of ten DAEC, nine EAEC strains, isolated from Mexican children with diarrhea, and one EAEC plus one commensal E. coli strains isolated from an adult with diarrhea and a healthy child, respectively. These genome sequences were compared to 85 E. coli genomes available in public databases. The EAEC and DAEC strains segregated into multiple different clades; however, six clades were heavily or exclusively comprised of EAEC and DAEC strains, suggesting a phylogenetic relationship between these two pathotypes. EAEC strains harbored the typical virulence factors under control of the activator AggR, but also several toxins, bacteriocins, and other virulence factors. DAEC strains harbored several iron-scavenging systems, toxins, adhesins, and complement resistance or Immune system evasion factors that suggest a pathogenic paradigm for this poorly understood pathotype. Several virulence factors for both EAEC and DAEC were associated with clinical presentations, not only suggesting the importance of these factors, but also potentially indicating opportunities for intervention. Our studies provide new insights into two distinct but related diarrheagenic organisms.

Diffusely Adherent Escherichia coli Strains Isolated from Healthy Carriers Suppress Cytokine Secretions of Epithelial Cells Stimulated by Inflammatory Substances.

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.

High prevalence of diarrheagenic Escherichia coli carrying toxin-encoding genes isolated from children and adults in southeastern Brazil

BackgroundDiarrheagenic Escherichia coli (DEC) are important bacterial causes of childhood diarrhea in Brazil, but its impact in adults is unknown. This study aimed at investigating DEC among children and adults living in endemic areas.MethodsA total of 327 stools specimens were collected from children (n = 141) and adults (n = 186) with diarrhea attending health centers. Diarrheagenic E. coli (DEC) were identified by their virulence genes (multiplex polymerase chain reaction) and HEp-2 cell adherence patterns.ResultsDEC were detected in 56 (40%) children and 74 (39%) adults; enteroaggregative E. coli (EAEC) (23%) was the most prevalent pathotype, followed by diffusely adherent E. coli (DAEC) (13%), and occurred at similar frequencies in both diarrheal groups. Atypical enteropathogenic E. coli (aEPEC) strains were recovered more frequently from children (6%) than from adults (1%). Twenty-six percent of the EAEC were classified as typical EAEC possessing aggR gene, and carried the aap gene. EAEC strains carrying aggR-aap-aatA genes were significantly more frequent among children than adults (p < 0.05). DAEC strains possessing Afa/Dr. genes were detected from children (10%) and adults (6%). EAEC and DAEC strains harboring genes for the EAST1 (astA), Pet, Pic, and Sat toxins were common in both diarrheal groups. The astA and the porcine AE/associated adhesin (paa) genes were found in most of aEPEC strains. High levels of resistance to antimicrobial drugs were found among DAEC and aEPEC isolates.ConclusionThe results show a high proportion of EAEC and DAEC carrying toxin-encoding genes among adults with diarrhea.

Molecular Detection of Diffusely Adherent Escherichia coli Strains Associated with Diarrhea in Shiraz, Iran

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.

Diarrheagenic Escherichia coli carrying supplementary virulence genes are an important cause of moderate to severe diarrhoeal disease in Mexico.

Diarrheagenic Escherichia coli (DEC) cause acute and persistent diarrhoea worldwide, but little is known about their epidemiology in Mexico. We determined the prevalence of bacterial enteropathogens in 831 children with acute diarrhoea over a four-year period in Yucatan, Mexico. Six DEC supplementary virulence genes (SVG), mainly associated with enteroaggregative E. coli (EAEC), were sought in 3100 E. coli isolates. DEC was the most common bacterial enteropathogen (28%), surpassing Salmonella (12%) and Shigella (9%). Predominant DEC groups were diffusely adherent E. coli (DAEC) (35%), EAEC (24%), and enteropathogenic E. coli (EPEC) (19%). Among children with DEC infections, 14% had severe illness mainly caused by EPEC (26%) and DAEC (18%); 30% had moderate diarrhoea mainly caused by DAEC (36%), mixed DEC infections (33%) and EAEC (32%). DAEC was most prevalent during spring, while ETEC, EAEC and EPEC predominated in summer. EAEC was more frequent in children 6–24 months old than in those younger than 6 months of age (P = 0.008, OR = 4.2, 95% CI, 1.3–13.9). The presence of SVG dispersin, (aatA), dispersin-translocator (aatA), enteroaggregative heat-stable toxin 1 (astA), plasmid encoded toxin (pet), cytolethal distending toxin (cdt) was higher in DEC than non-DEC strains, (36% vs 26%, P <0.0001, OR = 1.5, 95% CI, 1.3–1.8). 98% of EAEC-infected children harboured strains with SVG; 85% carried the aap-aatA gene combination, and 33% of these also carried astA. 28% of both EPEC and ETEC, and 6% of DAEC patients had strains with SVG. 54% of EPEC patients carried pet-positive strains alone or in combination with astA; only this DEC group harboured cdt-positive isolates. All ETEC patients carried astA- or astA-aap-positive strains. astA and aap were the most common SVG in DAEC (3% and 2%) and non-DEC strains (21% and 13%). DEC carrying SVG are an important cause of moderate to severe bacterial diarrhoea in Mexican children.

Persistent bloody diarrhoea without fever associated with diffusely adherent Escherichia coli in a young child

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.

Genotypic and phenotypic analysis of diarrheagenic Escherichia coli strains isolated from Brazilian children living in low socioeconomic level communities

BackgroundChildhood diarrheal diseases remain highly endemic in developing areas of Brazil. The importance of Escherichia coli among children with diarrhea in these areas was unknown. This study determined the prevalence of different E. coli categories in symptomatic and asymptomatic children from low socioeconomic level rural communities in southeastern Brazil.MethodsA total of 560 stool samples were collected from 141 children with diarrhea (< 10 years) and 419 apparently healthy controls who resided in 23 communities. E. coli isolates (n = 1943) were subjected to two multiplex PCRs developed for the detection of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and Shiga toxin-producing E. coli (STEC). Strains were also examined for the presence of EPEC, EAEC, and DAEC by assays of adhesion to HEp-2 cells and by hybridization with specific DNA probes.ResultsDiarrheagenic E. coli strains were isolated from 253 (45.2%) children, and were associated with diarrhea in children aged < 5 years (p < 0.001). EAEC (20.9%), DAEC (11.6%), EPEC (9.3%) were the most frequent pathotypes, followed by ETEC (2.7%), EIEC (0.5%), and STEC (0.2%). Depending of the assay, EPEC, EAEC, and DAEC (collectively termed enteroadherent E. coli) strains were isolated in 45% to 56% of diarrhea cases, a significantly higher incidence than in controls (P < 0.05). Individually, only DAEC showed significant association with diarrhea (p < 0.05), particularly in children aged 2–5 years.ConclusionThis study indicates that enteroadherent E. coli is an important cause of diarrhea in children living in low socioeconomic level communities in southeastern Brazil. Our results reveal that the PCR1 assay is an excellent tool for the identification of EAEC and DAEC.

Effect of diffusely adherent Escherichia coli strains isolated from diarrhoeal patients and healthy carriers on IL-8 secretion and tight junction barrier integrity of Caco-2 cells

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.

Phenotypic assay of adherent &lt;em&gt;E. coli&lt;/em&gt; strains using hep-2 cells on diarrheic children in Rivers State of Nigeria

In this paired case-control study of children with diarrhea in Rivers state, the association between HEp-2&#8211;adherent Escherichia coli strains and diarrhea was examined. Escherichia coli isolates from stool specimens of children with diarrhea were matched with controls and tested in HEp-2 cell adherence assay. A total of 266 E. coli strains (2 strains for every test subject) from both 83 children with diarrhea and 50 apparently healthy controls were examined for virulent traits using HEp-2 cells. Statistically significant (P&lt;0.05) adherent strains were obtained from the diarrheic children 18 (21.7%) as against 5(10%) from the control. While EPEC (3.6% vs 0%) and EAEC (10.8% vs 4%) strains were significantly associated with diarrhea (P&lt;0.05), EPEC was isolated only in children &lt;3 years old while EAEC was more distributed on the age ranges studied. There was no significant association of DAEC (7.2% vs 2%) strains isolated from the test subjects and the control group (P&gt;0.05%). High prevalence of parasites were seen on both groups although the diarrheal group had a statistical significant (26.5% vs 16%) prevalence (P&lt;0.05), this showed that intestinal parasites are also important factor in the etiology of diarrhea in this area. The parasites were mostly seen in older children from both groups and this might be attributed to their wandering, playing and eating habits. No mixed infection between parasites, parasite and adherent E. coli or between two adherents E. coli was observed. The adherent isolates showed poor sensitivity to traditional drugs like Ampicillin, Cotrimoxazole and Chloramphenicol, while Cefuroxime and Ceftazidime showed good sensitivity. These data suggest that EAEC may be a pathotype that is increasing in incidence as a cause of diarrhea in children in these areas.Keywords: Adherent E. coli (DEC); Enteropathogenic E. coli (EPEC); Enteroaggregative E. coli (EAEC); Diffusely adherent E. coli (DAEC); Mixed Infections; Intestinal Parasites

Patrones de adherencia de cepas de Escherichia coli Difusamente adherente (DAEC) provenientes de niños con y sin diarrea

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. To compare the adherence patterns between strains isolated from children with and without diarrhea. 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 identified by PCR (daaD gene). The pattern and adherence score in HEp-2 cell culture were evaluated, Actin polimerization was determined by fluorescence actin staining (FAS) and motility was evaluated by conventional microbiology methods. 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 significantly lower in diarrhea samples (p<0.05). However, actin polymerization was greater in diarrhea samples (60% vs. 17%). Motility test was positive in 60% of the diarrhea samples and in all control samples. Our findings suggest a difference between adherence patterns, actin polymerization and motility between DAEC strains corresponding to diarrhea and control groups. The significance of these results must be confirmed with a bigger number of strains and determining the presence of virulence genes in the strains.

Association of IL-8-inducing strains of diffusely adherent Escherichia coli with sporadic diarrheal patients with less than 5 years of age

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.

The secreted autotransporter toxin, Sat, functions as a virulence factor in Afa/Dr diffusely adhering Escherichia coli by promoting lesions in tight junction of polarized epithelial cells

Afa/Dr diffusely adhering Escherichia coli (DAEC) strains are responsible for urinary tract and intestinal infections. Both in intestine and kidney, the epithelial cells forming epithelium are sealed by junctional domains. We provide evidence that the Secreted autotransporter toxin, Sat, belonging to the subfamily of serine protease autotransporters of Enterobacteriaceae (SPATEs), acts as a virulence factor in Afa/Dr DAEC by promoting lesions in the tight junctions (TJs) of polarized epithelial Caco-2/TC7 cells. Southern blot analysis reveals that the prototype strains of the subclass-1 and subclass-2 typical Afa/Dr DAEC strains, hybridize with a sat probe. Using the wild-type IH11128 strain, the recombinant E. coli AAEC185 strain that expresses Sat, the recombinant E. coli that expresses both Dr adhesin and Sat, we report that Sat in monolayers of cultured enterocyte-like Caco-2/TC7 cells, induces rearrangements of the TJs-associated proteins ZO-1, ZO-3 and occludin, and increases the formation of domes as the result of an increase in the paracellular permeability without affecting the transepithelial electrical resistance of the cell monolayers. Moreover, we observe that Sat-induced disassembly of TJs-associated proteins is dependent on the serine protease motif. Finally, an analysis of the prevalence of the sat gene in three collections of Afa/Dr DAEC strains collected from the stools of children with and without diarrhoea, and from the urine of patients with urinary tract infection (UTI) shows that: (i) the sat gene is highly prevalent in UTI-associated Afa/Dr DAEC strains (88% positive), (ii) the sat gene is generally absent from Afa/Dr DAEC strains collected from the stools of children without diarrhoea (16% positive); whereas (iii) it is present in about half of the strains collected from the stools of children with diarrhoea (46% positive).

Epithelial Cells Secrete Interleukin‐8 in Response to Adhesion and Invasion of Diffusely Adhering Escherichia coli Lacking Afa/Dr Genes

Escherichia coli that sparsely adhere to human epithelial cells are known as diffusely adherent E. coli (DAEC), and the role of the Afa/Dr family of adhesins is now understood. Strains that do not possess Afa/Dr, however, comprise another group of DAEC, of which the pathogenicity remains unknown. The ability to induce interleukin-8 (IL-8) secretion from intestinal epithelial cells might be a feature of enterovirulent bacteria. We previously found that some Afa/Dr DAEC strains induce IL-8 by stimulating epithelial cells with flagella. The present study examines whether non-Afa/Dr DAEC can induce IL-8 in epithelial cells (HEp-2, INT407, and T84). Among 21 strains, 11 (52%; 11/21) induced as much IL-8 as high inducer strains of Afa/Dr DAEC. Adhesion did not significantly differ between high and low inducers; therefore diffuse adhesion alone is probably insufficient to induce IL-8. It was shown that IL-8 induction and the number of intracellular bacteria directly correlated. Wortmannin, an inhibitor of the phosphatidylinositol-3-phosphate kinase, reduced both intracellular bacteria and IL-8 secretion. Motile strains were significantly more prevalent among high (10/11) than low (4/10) inducers. However, 4 low invasive strains hardly induced IL-8 despite their motility. In conclusion, some non-Afa/Dr DAEC invoke the induction of high levels of inflammatory cytokines. Unlike Afa/Dr DAEC, however, non-Afa/Dr strains may require invasion to cause strong induction. These non-Afa/Dr high inducers can be enteropathogenic for the cytokine-inducing properties.

Diffusely adherent Escherichia coli strains expressing Afa/Dr adhesins (Afa/Dr DAEC): hitherto unrecognized pathogens

Diffusely adherent Escherichia coli (DAEC) strains are currently considered to constitute a putative sixth group of diarrheagenic E. coli. However, on the basis of their diffuse adherence to HEp-2 and HeLa cells, the detection of afa/dra/daa-related operons encoding this adherence phenotype, and the mobilization of decay-accelerating factor, both commensal and pathogenic strains can be classified as Afa/Dr DAEC isolates. Furthermore, strains associated with diarrheal diseases and strains causing extra-intestinal infections can also be identified as Afa/Dr DAEC strains. Although several cell signaling events that occur after epithelial cells have been infected by Afa/Dr DAEC have been reported, the pathophysiological processes that allow intestinal and extra-intestinal infections to develop are not fully understood. This review focuses on the genetic organization of the afa/dra/daa-related operons and on the virulence factors that trigger cellular responses, some of which are deleterious for the host cells. Finally, this review suggests future lines of research that could help to elucidate these questions.

Secreted autotransporter toxin produced by a diffusely adhering Escherichia coli strain causes intestinal damage in animal model assays

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.

Interleukin‐8 Secretion by Epithelial Cells Infected with Diffusely Adherent Escherichia coli Possessing Afa Adhesin‐Coding Genes

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.

Pathogenesis of Afa/Dr Diffusely AdheringEscherichia coli

Over the last few years, dramatic increases in our knowledge about diffusely adhering Escherichia coli (DAEC) pathogenesis have taken place. The typical class of DAEC includes E. coli strains 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/Dr(DAF) subclass) or carcinoembryonic antigen (CEA) (Afa/Dr(CEA) subclass). The atypical class of DAEC includes two subclasses of strains; the atypical subclass 1 includes E. coli strains 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 includes E. coli strains 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 beta(1) integrin 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.

Adhesins of Diffusely Adherent and Enteroaggregative Escherichia coli.

Epidemiological studies have implicated enteroaggregative Escherichia coli (EAEC) strains in acute and persistent diarrhea in children, in food-borne diarrhea outbreaks, and in traveler's diarrhea, and this group is recognized as an emerging pathotype of enteric disease. Diffusely adherent E. coli (DAEC) have been implicated as a cause of diarrhea, especially in children more than 2 years old, in both developing and developed countries. Although EAEC and DAEC strains appear to have different molecular equipment for attachment to host cell surfaces, identification and characterization of the gene clusters encoding adherence evidenced close relatedness between those determinants most frequently detected in isolates belonging to these two pathotypes of diarrheagenic E. coli. DAEC strains are a heterogeneous group of E. coli isolates, many of which express the related so-called Dr adhesins. The single designation is based on the identification of one similar cellular receptor for all these proteins. Although structurally different, they all recognize the Dr human blood group antigen on the decay-accelerating factor (DAF or CD55). These adhesins are encoded by a family of closely related operons, the first characterized and sequenced being the afa operon. Consequently, it has been suggested that this group of DAEC strains producing such adhesins be named the Afa/Dr DAEC family. Three distinct but closely related gene clusters coding for phenotypically and morphologically distinct aggregative adherence fimbriae (AAF) have been characterized. In each case, electron microscopy revealed that bacterial surfaces were surrounded by long, relatively flexible fimbrial structures.

Evidence for the presence of a type III secretion system in diffusely adhering Escherichia coli (DAEC)

Diffusely adhering Escherichia coli ( E. coli) strains (DAEC) represent a potential cause of diarrhoea in infants, and the detection of type three secretion system (TTSS) genes in DAEC would substantiate their pathogenic nature. In this work, four isolates of DAEC, recovered from stools of diarrhoeic children, were analysed by PCR, in order to detect the presence of TTSS genes. Primers targeted to the escC, escJ, escN and escV, some of the most conserved TTSS genes in enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC), were used in order to verify the occurrence of homologous genes in our DAEC isolates. By this approach, we were able to characterise DNA fragments corresponding to putative escJ and escN genes in all DAEC isolates. Furthermore, DNA fragments homologous to the escC and escV genes were also amplified from all isolates. Besides the similarity found among the DAEC esc homologues with EPEC and EHEC esc genes, the nucleotide sequence analysis of the flanking regions of the amplified DNA fragments suggests that the putative DAEC esc genes are organised in the same manner as observed in EPEC and in EHEC strains. The results described here provide strong evidence for the presence of a TTSS in the DAEC strains analysed, implicating a pathogenic nature of these isolates.

Impairments in enzyme activity and biosynthesis of brush border-associated hydrolases in human intestinal Caco-2/TC7 cells infected by members of the Afa/Dr family of diffusely adhering Escherichia coli.

Wild-type diffusely adhering Escherichia coli (DAEC) harbouring afimbrial adhesin (Afa) or fimbrial Dr and F1845 adhesins (Afa/Dr DAEC) apically infecting the human intestinal epithelial cells promote injuries in the brush border of the cells. We report here that infection by Afa/Dr DAEC wild-type strains C1845 and IH11128 in polarized human fully differentiated Caco-2/TC7 cells dramatically impaired the enzyme activity of functional brush border-associated proteins sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPP IV). Blockers of the transduction signal molecules, previously found to be active against the Afa/Dr DAEC-induced cytoskeleton injury, were inactive against the Afa/Dr-induced decrease in sucrase enzyme activity. In parallel, Afa/Dr DAEC infection promotes the blockade of the biosynthesis of SI and DPP IV without affection enzyme stability. The observation that no changes occurred in mRNA levels of SI and DPP IV upon infection suggested that the decrease in biosynthesis probably resulted from a decrease in the translation rate. When the cells were infected with recombinant E. coli strains expressing homologous adhesins of the wild-type strains, neither a decrease in sucrase and DPP IV enzyme activities nor an inhibition of enzyme biosynthesis were observed. In conclusion, taken together, these data give new insights into the mechanisms by which the wild-type Afa/Dr DAEC strains induce functional injuries in polarized fully differentiated human intestinal cells. Moreover, the results revealed that other pathogenic factor(s) distinct from the Afa/Dr adhesins may play(s) a crucial role in this mechanism of pathogenicity.