Phenotypic and genotypic characteristics of Escherichia coli strains of non-enteropathogenic E-coli (EPEC) serogroups that carry eae and lack the EPEC adherence factor and shiga toxin DNA probe sequences

This study was conducted to characterize the virulence potential of 59 Escherichia coli strains carrying eae and lacking the enteropathogenic E. coli adherence factor and Shiga toxin probe sequences. in hybridization studies, all strains carried the locus of enterocyte effacement (LEE)-associated DNA sequences. of the other 15 virulence DNA sequences tested, hly was the most frequent (44.1%); 17 combinations of these sequences were found, but strains carrying eae only (eae profile) were the most frequent (35.6%). Except for 1 cytodetaching strain, all others adhered to HeLa and Caco-2 cells, most of which (similar to 75.0%) showed variations of the localized adherence pattern. Actin accumulation was detected in 75.9% of the nondetaching strains. Most strains had LEE, probably inserted in pheU (49.2%), and presented a nontypeable intimin (83.1%). Translocated intimin receptor-derived DNA sequences correlated with enteropathogenic and enterohemorrhagic E. coli in 61.0% and 32.0% of the strains, respectively. Thirty-five different serotypes were found. Only strains with the eae profile were associated with diarrhea (P = .039).Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilUniv São Paulo, Inst Butantan, Lab Especial Microbiol, São Paulo, BrazilUniv São Paulo, Inst Adolfo Lutz, Secao Bacteriol, São Paulo, BrazilUniv São Paulo, Hosp Clin, Inst Crianca, São Paulo, BrazilUniv Estado Rio de Janeiro, Dept Microbiol, Rio de Janeiro, BrazilUniv London Imperial Coll Sci Technol & Med, Dept Biochem, London, EnglandUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilWeb of Scienc

An agonist sensitive, quick and simple cell-based signaling assay for determination of ligands mimicking prostaglandin E2 or E1 activity through subtype EP1 receptor: Suitable for high throughput screening

<p>Abstract</p> <p>Background</p> <p>Conventionally the active ingredients in herbal extracts are separated into individual components, by fractionation, desalting, and followed by high-performance liquid chromatography (HPLC). In this study we have tried to directly screen water-soluble fractions of herbs with potential active ingredients before purification or extraction. We propose that the herbal extracts mimicking prostaglandin E₁(PGE₁) and E₂(PGE₂) can be identified in the water-soluble non-purified fraction. PGE₁is a potent anti-inflammatory molecule used for treating peripheral vascular diseases while PGE₂is an inflammatory molecule.</p> <p>Methods</p> <p>We used cell-based assays (CytoFluor multi-well plate reader and fluorescence microscopy) in which a calcium signal was generated by the recombinant EP₁receptor stably expressed in HEK293 cells (human embryonic kidney). PGE₁and PGE₂were tested for their ability to generate a calcium signal. Ninety-six water soluble fractions of Treasures of the east (single Chinese herb dietary supplements) were screened.</p> <p>Results</p> <p>After screening, the top ten stimulators were identified. The identified herbs were then desalted and the calcium fluorescent signal reconfirmed using fluorescence microscopy. Among these top ten agonists identified, seven stimulated the calcium signaling (1-40 μM concentration) using fluorescence microscopy.</p> <p>Conclusions</p> <p>Fluorescence microscopy and multi-well plate readers can be used as a target specific method for screening water soluble fractions with active ingredients at a very early stage, before purification. Our future work consists of purifying and separating the active ingredients and repeating fluorescence microscopy. Under ordinary circumstances we would have to purify the compounds first and then test all the extracts from 96 herbs. Conventionally, for screening natural product libraries, the procedure followed is the automated separation of all constituents into individual components using fractionation and high performance liquid chromatography. We, however, demonstrated that the active ingredients of the herbal extracts can be tested before purification using an agonist sensitive, quick and simple cell-based signaling assay for ligands mimicking the agonists, PGE₁and PGE₂.</p