Aspectos celulares, moleculares e ultraestruturais da interação de Escherichia coli enteroagregativa com células não intestinais e intestinais humanas in vitro e epitélio intestinal de coelhos ex vivo

Abstract

Enteroaggregative Escherichia coli (EAEC) are an emerging pathotype associated with cases of acute or persistent diarrhea in children and adults in developed and emerging countries. It causes persistent diarrhea in HIV positive patients and it is the second most common cause of traveler s diarrhea. The aggregative adherence (AA) pattern characterized by arrangement in stacked brick monolayer HEp-2 (human larynx carcinoma) is related to the presence of a plasmid aggregative adherence (pAA) of 60 MDa. The hypothetical pathogenesis is represented by the heterogeneity of the category that covers a variety of putative virulence factors involved in the processes of colonization, cell adhesion and cytotoxicity. The present study aimed to investigate the ability of producing the standard AA by EAEC strains in HEp-2, Caco-2 (human colorectal adenocarcinoma) and T84 (human intestinal colon carcinoma), the correlation between AA and pAA in each strain in addition to the interaction of EAEC strains using in vitro organ culture model (IVOC). For the first two objectives were used seventeen EAEC strains (10 from children with acute diarrhea and seven from children without diarrhea). For the last were employed five EAEC strains from children with acute diarrhea. The adhesion assays were performed with strains co-incubated with cell lines under study for a period of 3 h and 6 h. Among the 17 strains EAEC 29.4%, 41.1% and 82.3% showed AA at 3 h in cell lines HEp-2, Caco-2 and T84, respectively. In 6 h, 52.9% EAEC strains exhibited AA in HEp-2 and Caco-2 and 100% strains exhibited AA in T84. As the line T84 has phenotypic characteristics of colon cells and Caco-2 of the cells of the small intestine, data suggested that EAEC may have a higher tropism for the human colon. PCR for detection of pAA showed 29.4%, 41.1% and 52.9% of the strains as pAA-positive showing the AA pattern in HEp-2, Caco-2 and T84 cells, simultaneously, in tests of 3 h. In 6 h, 47%, 52.9% and 70.5% of the strains pAA + exhibited AA pattern in HEp-2, Caco-2 and T84, respectively. The in vitro adhesion assays using cultured tissue were performed with EAEC strains co-incubated with intestinal fragments of ileum and colon over a period of 6 h. Light microscopy (LM) revealed two strains more adherent to the colon, one to the ileum and two to both. The scanning electron microscopy (SEM) showed three strains more adherent to the colon, one to the ileum and another to both. Bacterial aggregates on the epithelium or overlapping the mucus were observed by LM and SEM. LM assays also showed morphological changes such as cellular extrusion, structural disorganization and destruction of the villi. The results showed that the line T84 proved to be more susceptible to aggregative pattern formation, as well as the adherence of strains AA / pAA +, which was ratified by the higher degree of adherence to the colonic mucosa in the IVOC. Finally, data indicated that these models are more effective for elucidating the pathogenicity of these strains.