Atypical enteropathogenic Escherichia coli (aEPEC) has been associated with infantile diarrhea in many countries. The clonal structure of aEPEC is the object of active investigation but few works have dealt with its genetic relationship with other diarrheagenic E. coli (DEC). This study aimed to evaluate the genetic relationship of aEPEC with other DEC pathotypes. The phylogenetic relationships of DEC strains were evaluated by multilocus sequence typing. Genetic diversity was assessed by pulsed-field gel electrophoresis (PFGE). The phylogram showed that aEPEC strains were distributed in four major phylogenetic groups (A, B1, B2 and D). Cluster I (group B1) contains the majority of the strains and other pathotypes [enteroaggregative, enterotoxigenic and enterohemorrhagic E. coli (EHEC)]; cluster II (group A) also contains enteroaggregative and diffusely adherent E. coli; cluster III (group B2) has atypical and typical EPEC possessing H6 or H34 antigen; and cluster IV (group D) contains aEPEC O55:H7 strains and EHEC O157:H7 strains. PFGE analysis confirmed that these strains encompass a great genetic diversity. These results indicate that aEPEC clonal groups have a particular genomic background--especially the strains of phylogenetic group B1--that probably made possible the acquisition and expression of virulence factors derived from non-EPEC pathotypes.

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BackgroundDiarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains.Methods and FindingsRecombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains.ConclusionThe developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

BackgroundEnteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens.MethodologyFirst, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT) was developed and tested with the same collection of bacterial isolates.Principal findingsEspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world.ConclusionRALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.

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Hybrid-pathogenic Escherichia coli represent an important group of strains associated with intestinal and extraintestinal infections. Recently, we described strain UPEC-46, a uropathogenic/enteroaggregative E. coli (UPEC/EAEC) strain presenting the aggregative adherence (AA) pattern on bladder and colorectal epithelial cells mediated by aggregate-forming pili (AFP). However, the role of AFP and other uninvestigated putative fimbriae operons in UPEC-46 pathogenesis remains unclear. Thus, this study evaluated the involvement of AFP and other adhesins in uropathogenicity and intestinal colonization using different in vitro and in vivo models. The strain UPEC-46 was able to adhere and invade intestinal and urinary cell lines. A library of transposon mutants also identified the involvement of type I fimbriae (TIF) in the adherence to HeLa cells, in addition to colorectal and bladder cell lines. The streptomycin-treated mouse in vivo model also showed an increased number of bacterial counts in the colon in the presence of AFP and TIF. In the mouse model of ascending urinary tract infection (UTI), AFP was more associated with kidney colonization, while TIF appears to mediate bladder colonization. Results observed in in vivo experiments were also confirmed by electron microscopy (EM) analyses. In summary, the in vitro and in vivo analyses show a synergistic role of AFP and TIF in the adherence and colonization of intestinal and urinary epithelia. Therefore, we propose that hybrid E. coli strains carrying AFP and TIF could potentially cause intestinal and urinary tract infections in the same patient.

Título daPadronização e avaliação de PCR multiplex para o diagnóstico de Escherichia coli enteroagregativa típica e atípica.