Enteroaggregative Escherichia Coli Strains Research Articles

Atypical enteropathogenic Escherichia coli secretes plasmid encoded toxin.

Plasmid encoded toxin (Pet) is a serine protease originally described in enteroaggregative Escherichia coli (EAEC) prototype strain 042 whose entire characterization was essentially obtained from studies performed with the purified toxin. Here we show that Pet is not exclusive to EAEC. Atypical enteropathogenic Escherichia coli (aEPEC) strains, isolated from diarrhea cases, express Pet and its detection in supernatants of infected HEp-2 cells coincides with the appearance of cell damage, which, in turn, were similar to those described with purified Pet. Pet secretion and the cytotoxic effects are time and culture medium dependent. In presence of DMEM supplemented with tryptone cell rounding and detachment were observed after just 5 h of incubation with the bacteria. In the absence of tryptone, the cytotoxic effects were detected only after 24 h of infection. We also show that, in addition to the prototype EAEC, other pet+ EAEC strains, also isolated from diarrhea cases, induce cellular damage in the same degree as the aEPEC. The cytotoxic effects of EAEC and aEPEC strains were significantly reduced in the presence of a serine protease inhibitor or anti-Pet IgG serum. Our results show a common aspect between the aEPEC and EAEC and provide the first evidence pointing to a role of Pet in aEPEC pathogenesis.

Whole genome sequence comparison of vtx2-converting phages from Enteroaggregative Haemorrhagic Escherichia coli strains.

BackgroundEnteroaggregative Haemorrhagic E. coli (EAHEC) is a new pathogenic group of E. coli characterized by the presence of a vtx2-phage integrated in the genomic backbone of Enteroaggregative E. coli (EAggEC). So far, four distinct EAHEC serotypes have been described that caused, beside the large outbreak of infection occurred in Germany in 2011, a small outbreak and six sporadic cases of HUS in the time span 1992–2012. In the present work we determined the whole genome sequence of the vtx2-phage, termed Phi-191, present in the first described EAHEC O111:H2 isolated in France in 1992 and compared it with those of the vtx-phages whose sequences were available.ResultsThe whole genome sequence of the Phi-191 phage was identical to that of the vtx2-phage P13374 present in the EAHEC O104:H4 strain isolated during the German outbreak 20 years later. Moreover, it was also almost identical to those of the other vtx2-phages of EAHEC O104:H4 strains described so far. Conversely, the Phi-191 phage appeared to be different from the vtx2-phage carried by the EAHEC O111:H21 isolated in the Northern Ireland in 2012.The comparison of the vtx2-phages sequences from EAHEC strains with those from the vtx-phages of typical Verocytotoxin-producing E. coli strains showed the presence of a 900 bp sequence uniquely associated with EAHEC phages and encoding a tail fiber.ConclusionsAt least two different vtx2-phages, both characterized by the presence of a peculiar tail fiber-coding gene, intervened in the emergence of EAHEC. The finding of an identical vtx2-phage in two EAggEC strains isolated after 20 years in spite of the high variability described for vtx-phages is unexpected and suggests that such vtx2-phages are kept under a strong selective pressure.The observation that different EAHEC infections have been traced back to countries where EAggEC infections are endemic and the treatment of human sewage is often ineffective suggests that such countries may represent the cradle for the emergence of the EAHEC pathotype. In these regions, EAggEC of human origin can extensively contaminate the environment where they can meet free vtx-phages likely spread by ruminants excreta.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-574) contains supplementary material, which is available to authorized users.

Participation of integrin α5β1 in the fibronectin-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrin α5β1, in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin α5β1; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrin α5β1. To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin α5β1 to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin α5 shRNA. Altogether, our data support the involvement of integrin α5β1 in the fibronectin-mediated EAEC binding to intestinal cells.

Diarrheagenic enteroaggregative Escherichia coli causing urinary tract infection and bacteremia leading to sepsis

We report a case of a 55-year-old immunocompromised female who presented to the emergency department with severe diarrhea and vomiting following travel to the Philippines. Stool bacteriology revealed a mixed infection involving an enteropathogenic Escherichia coli and two distinct strains of enteroaggregative Escherichia coli (EAEC). During hospitalization, urine and blood culture tested positive for one of the diarrheagenic EAEC strains, necessitating urinary catheterization, intensive care, and antimicrobial treatment with trimethoprim-sulfamethoxazole, followed by meropenem. Although known to occasionally cause urinary tract infections, EAEC have not been previously associated with sepsis. Our report highlights the potential of EAEC to cause severe extraintestinal infections.

The micronutrient zinc inhibits EAEC strain 042 adherence, biofilm formation, virulence gene expression, and epithelial cytokine responses benefiting the infected host

Enteroaggregative Escherichia coli (EAEC) is a major pathogen worldwide, associated with diarrheal disease in both children and adults, suggesting the need for new preventive and therapeutic treatments. We investigated the role of the micronutrient zinc in the pathogenesis of an E. coli strain associated with human disease. A variety of bacterial characteristics—growth in vitro, biofilm formation, adherence to IEC-6 epithelial cells, gene expression of putative EAEC virulence factors as well as EAEC-induced cytokine expression by HCT-8 cells—were quantified. At concentrations (≤ 0.05 mM) that did not alter EAEC growth (strain 042) but that are physiologic in serum, zinc markedly decreased the organism’s ability to form biofilm (P < 0.001), adhere to IEC-6 epithelial cells (P < 0.01), and express putative EAEC virulence factors (aggR, aap, aatA, virK) (P < 0.03). After exposure of the organism to zinc, the effect on virulence factor generation was prolonged (>3 h). Further, EAEC-induced IL-8 mRNA and protein secretion by HCT-8 epithelial cells were significantly reduced by 0.05 mM zinc (P < 0.03). Using an in vivo murine model of diet-induced zinc-deficiency, oral zinc supplementation (0.4 µg/mouse daily) administered after EAEC challenge (1010 CFU/mouse) significantly abrogated growth shortfalls (by >90%; P < 0.01); furthermore, stool shedding was reduced (days 9–11) but tissue burden of organisms in the intestine was unchanged. These findings suggest several potential mechanisms whereby physiological levels of zinc alter pathogenetic events in the bacterium (reducing biofilm formation, adherence to epithelium, virulence factor expression) as well as the bacterium’s effect on the epithelium (cytokine response to exposure to EAEC) to alter EAEC pathogenesis in vitro and in vivo. These effects may help explain and extend the benefits of zinc in childhood diarrhea and malnutrition.

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.

Characteristics of enteroaggregative Escherichia coli isolated from healthy carriers and from patients with diarrhoea

The aim of this study was to compare the virulence characteristics and phylogenetic features of enteroaggregative Escherichia coli (EAEC) strains from adults with and without diarrhoea and to search for associations between the analysed genes and carrier or diarrhoeagenic strains, respectively. Faecal samples of 487 healthy humans were screened for EAEC strains and compared with isolates from diarrhoeal patients. Virulence and virulence-associated gene typing, serotyping, multilocus sequence typing and antibiotic susceptibility testing were performed for characterization of the isolates. Characteristics significantly linked to carrier strains or to diarrhoeagenic strains were determined. From 487 stool samples, 24 EAEC strains were obtained. Comparison with strains originating from diseased persons showed a statistically significant association of the genes sat (P = 0.002) and agg3C (P = 0.0139) with the carrier strains, and of pCVD432 (P = 0.0001), aap (P = 0.003), aggR (P = 0.0048) and air (P = 0.031) with the diarrhoeagenic strains. Our study indicates that a certain subset of EAEC is unrelated to diarrhoea, for which sat and agg3C may be markers. Our results further suggest that diarrhoeagenic EAEC strains are distinguishable from carrier strains and suggest that, in addition to well-established markers such as pCVD432 and aggR, aap and air may be useful additional markers to define EAEC as an aetiological agent of diarrhoea in adults.

Behavior of enteroaggregative Escherichia coli, non-O157-shiga toxin-producing E. coli, enteroinvasive E. coli, enteropathogenic E. coli and enterotoxigenic E. coli strains on mung bean seeds and sprout

The behavior of enteroaggregative Escherichia coli (EAEC), non-O157 shiga toxin-producing E. coli (non-O157-STEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) on mung bean seeds at 25±2°C and during germination and sprouting of mung bean seeds at 20±2° and 30±2°C and on mung bean sprouts at 3±2°C was determined. When mung bean seeds were inoculated with EAEC, non-O157 STEC, EIEC, EPEC or ETEC strains, all these diarrheagenic E. coli pathotypes (DEPs) survived at least 90days on mung bean seeds at 25±2°C. All DEPs grew during germination and sprouting of seeds, reaching counts of approximately 5 Log and 7 Log CFU/g after 2days at 20±2° and 30±2°C, respectively. However, when the sprouts were inoculated after 1day of seeds germination and stored at 20±2° or 30±2°C, no growth was observed for any DEPs during sprouting at 20±2°C per 9 d; however, a significant increase in the concentration of DEPs of approximately 0.7 log CFU/g was observed during sprouting at 30±2°C after 1day of sprout contamination. Refrigeration reduced the number of viable DEPs strains on sprouts after 10days in storage; nevertheless, these decreases have no practical significance in the safety of the sprouts.

The role of entero-aggregative Escherichia coli (EAEC) strains on diarrheic children in some southern States of Nigeria

The role of entero-aggregative Escherichia coli (EAEC) strains on diarrheic children in some southern States of Nigeria was carried out in this paired control study. 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 800 E. coli strains (2 strains for every test subject) from both 250 children with diarrhea and 150 apparently healthy controls were examined for aggregative adhesion using HEp-2 cells. Statistically significant (P<0.05) EAEC strains were obtained from the diarrheic children 40.6% as against 20% from the control. In this study, Bayelsa State recorded the highest isolate with 21.4 vs 12%, followed by Rivers State (10.8% vs 4.0%), then Imo State the least (8.4% vs 4.0%). Children within 4-8 years age range recorded the highest prevalence in the States studied. High prevalence of intestinal parasites were seen on both groups although the diarrheal group had a statistical significant (91% vs 36%) prevalence (P<0.05). The parasites were Giardia lamblia, Ascaris lumbricoides , hookworm and Trichuris trichuria . This showed that intestinal parasites are also important factor in the etiology of diarrhea in this area. There was mixed infection between parasites and EAEC in 5 test subjects in Bayelsa State, but none in the other States and also none from the control. The entero-aggregative E. coli showed marked resistance to conventional antimicrobial agents like cotrimoxazole, ampicillin and chloramphenicol with 80.3%, 81.2% and 61.6% respectively, while the cephalosporins: cefuroxime and ceftazidime showed low resistance with 3.3% and 4.3% respectively, indicating that the cephalosporins will be a good choice for the empirical treatment of bacterial infectious diarrhea in this region. Keywords: HEp-2 cells, Intestinal parasites, Mixed infection, Antimicrobial agents, Cephalosporins

Genotypic and Phenotypic Characterisation of Enteroaggregative Escherichia coli from Children in Rio de Janeiro, Brazil

Enteroaggregative Escherichia coli (EAEC) is a significant cause of diarrhoeal illness in both children and adults. Genetic heterogeneity and recovery of EAEC strains from both healthy and diseased individuals complicates our understanding of EAEC pathogenesis. We wished to establish if genetic or phenotypic attributes could be used to distinguish between strains asymptomatically colonising healthy individuals and those which cause disease. Genotypic screening of a collection of twenty four EAEC isolates from children with and without diarrhoea revealed no significant differences in the repertoire of putative virulence factors present in either group of strains. In contrast, EAEC strains from phylogroup A were more strongly associated with asymptomatic groups whereas strains from phylogroup D were more associated with cases of diarrhoea. Phenotypic screening revealed no differences in the ability of strains from either cohort of children to form biofilms, to adhere to and invade cells in tissue culture or to cause disease in the Caenorhabditis elegans model of infection. However, the latter assay did reveal significant reduction in nematode killing rates when specific virulence factors were deleted from human pathogenic strains. Our results suggest that current models of infection are not useful for distinguishing avirulent from pathogenic strains of EAEC but can be useful in studying the effect of specific virulence factors.

Enteroaggregative Escherichia coli strain in a novel weaned mouse model: exacerbation by malnutrition, biofilm as a virulence factor and treatment by nitazoxanide

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a common cause of diarrhoea in healthy, malnourished and immune-deficient adults and children. There is no reproducible non-neonatal animal model for longitudinal studies of disease mechanism or therapy. Using two strains of human-derived EAEC to challenge weaned C57BL/6 mice, we explored an in vivo model of EAEC infection in mice, in which disease was monitored quantitatively as the growth rate, stool shedding and tissue burden of organisms; nutritional status was varied, and a new class of therapeutics was assessed. A single oral challenge of EAEC strain 042 resulted in significant growth shortfalls (5-8 % of body weight in 12 days), persistent shedding of micro-organisms in stools [>10(3.2) c.f.u. (10 mg stool)(-1) for at least 14 days] and intestinal tissue burden [~10(3) c.f.u. (10 mg tissue)(-1) detectable up to 14 days post-challenge]. Moderate malnourishment of mice using a 'regional basic diet' containing 7 % protein and reduced fat and micronutrients heightened all parameters of infection. Nitazoxanide in subMIC doses, administered for 3 days at the time of EAEC challenge, lessened growth shortfalls (by >10 % of body weight), stool shedding [by 2-3 logs (10 mg stool)(-1)] and tissue burden of organisms (by >75 % in the jejunum and colon). Thus, weaned C57BL/6 mice challenged with EAEC is a convenient, readily inducible model of EAEC infection with three highly quantifiable outcomes in which disease severity is dependent on the nutritional status of the host, and which is modifiable in the presence of inhibitors of pyruvate ferredoxin oxidoreductase such as nitazoxanide.

The Role of Peroxisome Proliferator-Activated Receptor γ in Immune Responses to Enteroaggregative Escherichia coli Infection

BackgroundEnteroaggregative Escherichia coli (EAEC) is recognized as an emerging cause of persistent diarrhea and enteric disease worldwide. Mucosal immunity towards EAEC infections is incompletely understood due in part to the lack of appropriate animal models. This study presents a new mouse model and investigates the role of peroxisome proliferator-activated receptor gamma (PPARγ) in the modulation of host responses to EAEC in nourished and malnourished mice.Methods/Principal FindingsWild-type and T cell-specific PPARγ null C57BL/6 mice were fed protein-deficient diets at weaning and challenged with 5×109cfu EAEC strain JM221 to measure colonic gene expression and immune responses to EAEC. Antigen-specific responses to E. coli antigens were measured in nourished and malnourished mice following infection and demonstrated the immunosuppressive effects of malnutrition at the cellular level. At the molecular level, both pharmacological blockade and deletion of PPARγ in T cells resulted in upregulation of TGF-β, IL-6, IL-17 and anti-microbial peptides, enhanced Th17 responses, fewer colonic lesions, faster clearance of EAEC, and improved recovery. The beneficial effects of PPARγ blockade on weight loss and EAEC clearance were abrogated by neutralizing IL-17 in vivo.ConclusionsOur studies provide in vivo evidence supporting the beneficial role of mucosal innate and effector T cell responses on EAEC burden and suggest pharmacological blockade of PPARγ as a novel therapeutic intervention for EAEC infection.

Prevalence of enteroaggregative Escherichia coli and its virulence-related genes in a case–control study among children from north-eastern Brazil

Enteroaggregative Escherichia coli (EAEC) is an important agent that causes endemic and epidemic diarrhoeal diseases worldwide. Several EAEC virulence-related genes (VRGs) have been described but their role in the clinical outcome of infection is not completely defined. This study investigated the prevalence of EAEC and potential associations of its VRGs with risk of or protection from diarrhoeal diseases in children from urban communities in north-eastern Brazil. The case-control study included 166 children, who had their stools evaluated for the EAEC diagnostic genes (aaiC and aatA) using PCR. Positive samples were further analysed by multiplex PCR and identified 18 VRGs. EAEC was found in the same proportion in both groups (41%). The plasmid-borne gene encoding a hexosyltransferase homologue (capU) was the most frequently detected (89.6%), followed by dispersin protein (aap, 58.2%) and EAEC HilA homologue (eilA, 57.8%). The AAF/III fimbrial subunit (agg3A) gene was observed at lower frequency (1.5%). Plasmid-encoded toxin (pet) or AAF/II fimbrial subunit (aafA) was associated significantly with disease. AAF/IV fimbrial subunit (agg4A) or hypothetical plasmid-encoded haemolysin (orf61) was detected significantly more in controls than in children with diarrhoea. In addition, one set of genes in combination, aaiC and agg3/4C but lacking agg4A and orf61, was associated with diarrhoea cases; and another one, orf61 in the absence of pet and aafA, was correlated with control children. These data confirm a high prevalence, endemicity and heterogeneity of EAEC strains in the developing urban areas of north-eastern Brazil. Statistical correlation between cases and controls was seen with either isolated or combined sets of genes, suggesting that the pathophysiology of EAEC infection involves a complex and dynamic modulation of several VRGs.

Epidemiological Relationship of EnterotoxigenicEscherichia coliand EnteroaggregativeE. coliIsolated from Patients with Diarrhea in Seoul

Of total 1,438 specimens of patients with diarrhea in Seoul, 2011, 217 samples (15%) were found pathogenic Escherichia coli that included 192 strains (89%) of enterotoxigenic E. coli (ETEC) and enteroaggregative E. coli (EAEC). The highest isolation rate for ETEC and EAEC was found in August and September. Sixty two pathogenic E. coli strains (34 ETEC and 28 EAEC strains) were selected from 175 strains (94 ETEC and 81 EAEC strains) isolated in August and September. Of 94 strains characterized for ETEC phenotype, 76 (81%) expressed heat-stable toxin (ST) only. Antimicrobial susceptibility test was carried out by using sixteen types of antibiotics. A high level of antimicrobial resistance to tetracycline (57%), ampicillin and ticarcillin (54%) was observed among EAEC isolates while the highest resistance rate of ETEC was found for nalidixic acid (47%), followed by tetracycline (32%). As to the antimicrobial susceptibility test, EAEC showed the complicated multi-drug resistant patterns in which the resistance was higher than ETEC. Pulsed-field gel electrophoresis (PFGE) was carried out to examine the genetic relatedness among ETEC and EAEC isolates. Except for 11 strains, 51 strains were divided by eight pulsotypes. In PFGE analysis, isolates from foodborne disease outbreaks in August and September 2011 showed close relation.

Rapid Detection Method for Enteroaggregative &amp;lt;i&amp;gt;Escherichia coli&amp;lt;/i&amp;gt; Using Simple Clump Formation and Aggregative Assay

Enteroaggregative Escherichia coli (EAggEC) strains cause the persistent diarrhea in infants and compromised hosts in developing countries. These strains are currently defined as E. coli that adheres to HEp-2 cells in an aggregative adherence (AA) pattern. In this study, we compared 4 different rapid methods for the detection of EAggEC using a PCR assay, clump formation test, glass slide adherence assay, and the HEp-2 cell adherence assay. Out of 683 E. coli strains isolated from diarrheal stool samples, we detected 17 aggR and/or clump-positive strains, and identified 2 aggR-positive, clump-negative strains and 2 aggR-negative, clump-positive strains. All the aggR positive and clump positive strains also showed positive results in glass slide adherence and HEp-2 cell adherence assays. From all these results, we suggest the following procedure for the rapid identification of EAggEC strains: first, screen E. coli strains with the clump formation test and subsequently perform the glass slide adherence assay to observe AA for confirmation.

Enteroaggregative Escherichia coli, a heterogenous, underestimated and under-diagnosed E. coli pathotype in Iran.

The main features of enteroaggregative Escherichia coli (EAEC) pathogenesis include attachment of bacteria to the intestinal mucosa, production of various toxins and cytotoxins, and stimulation of mucosal inflammation. 'Virulence' genes encode these features. Comparison of different EAEC isolates has shown that the virulence gene content of these isolates varies considerably. The heterogeneity of EAEC strains was concluded from the results obtained from the volunteer as well as other studies. Although the underlying mechanism behind the apparent increase in O104:H4 virulence is not known, several bacterial factors have been implicated. In this review, the known virulence factors involved in pathogenesis of EAEC pathotype are summarized.

Inflammatory diarrhea due to enteroaggregative Escherichia coli: evidence from clinical and mice model studies

BackgroundThis study was conducted to determine the role of enteroaggregative Escherichia coli (EAEC) in inflammatory diarrhea among hospitalized patients in Kolkata. The inflammatory pathogenesis of EAEC was established in mice model and histopathological studies. Presence of fecal leucocytes (FLCs) can be suspected for EAEC infection solely or as a mixed with other enteric pathogens.MethodsActive surveillance was conducted for 2 years on 2 random days per week with every 5th patient admitted to the Infectious Diseases Hospital (IDH). Diarrheal samples were processed by conventional culture, microscopy, ELISA and molecular methods. Two EAEC isolated as sole pathogens were examined in mice after induced intestinal infection. The intestinal tissue samples were processed to analyze the histological changes.ResultsOf the 2519 samples screened, fecal leucocytes, erythrocytes and occult blood were detected in 1629 samples. Most of the patients had acute watery diarrhea (75%) and vomiting (78%). Vibrio cholerae O1 was the main pathogen in patients of 5–10 years age group (33%). Shigellosis was more in children from 2–5 years of age (19%), whereas children <2 years appeared to be susceptible for infection caused by EAEC (16%). When tested for the pathogenicity, the EAEC strains colonized well and caused inflammatory infection in the gut mucosa of BALB/C mice.ConclusionThis hospital-based surveillance revealed prevalence of large number of inflammatory diarrhea. EAEC was the suspected pathogen and <2 years children appeared to be the most susceptible age group. BALB/C mice may be a suitable animal model to study the EAEC-mediated pathogenesis.

Enteroaggregative Escherichia coli quantification in children stool samples using quantitative PCR

Enteroaggregative Escherichia coli (EAEC) is a common cause of infectious diarrhea, especially in children living in poor-resource countries. In this article, we present a SYBR Green-based real-time polymerase chain reaction (qPCR) method for quantitative detection of EAEC in DNA directly extracted from human stool samples. To test the proposed qPCR system, we examined specificity, sensitivity, repeatability, and also the degree of DNA extraction efficiency using EAEC strain 042 spiked into EAEC-free stool sample. The specificity of this assay was proved using six strains of EAEC, seven strains of other E. coli types, and one strain of Shigella. The detection limit of qPCR was 67 CFU/reaction. In naturally infected stool samples, we found EAEC in quantities varying from 6.7 × 10(5) to 2 × 10(9 ) CFU/g of feces. We could not detect any reduction after stool DNA extraction for the amounts of 10(7) and 10(6) CFU/mL of spiked EAEC. This qPCR assay is simple, rapid, reproducible, sensitive, specific, and allows rapid EAEC quantification to be used in a variety of further EAEC studies. This new quantitative method provides a relatively simple means to quantify EAEC, which will likely be key to understanding the pathophysiology and impact of EAEC infection.

Characterization of the AggR Regulon in Enteroaggregative Escherichia coli

AggR is a transcriptional regulator of enteroaggregative Escherichia coli (EAEC) and has been proposed as the defining factor for typical EAEC strains. Expression of multiple putative virulence factors, including the aggregative adherence fimbriae (AAF), dispersin, the dispersin translocator Aat, and the Aai type VI secretion system, have been found to be regulated by AggR. Here, we confirm the existence of at least 44 AggR-regulated genes using DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR); these genes include chromosomal and plasmid-borne loci and 19 previously unsuspected genes. Two previously uncharacterized virulence plasmid-encoded open reading frames (ORFs) (designated ORF3 and ORF4) exhibit significant identity with isoprenoid biosynthesis genes of Bacteria and Archaea. The predicted ORF4 product is closely related to isopentenyl isomerase (IDI) enzymes, whereas the predicted product of the adjacent ORF3 exhibits an aspartate-rich region that is common among trans-isoprenyl phosphate synthases. We show that mutations in these ORFs confer changes in bacterial surface properties. AggR coordinately controls expression of a large number of EAEC genes.

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