Caracterização molecular da formação de biofilme por Escherichia coli enteropatogênica (EPEC) O119

• Autor(a) principal: Flores, Maricelia Navarro Pinheiro [UNIFESP]
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UNIFESP
• Texto Completo: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=7352879; https://repositorio.unifesp.br/handle/11600/59351
• topic: EPEC; Mutagênese; Biofilm; EPEC; Biofilme

EPEC can be categorized into two subgroups, typical (tEPEC) and atypical (aEPEC), based on the presence or absence of the EPEC adherence factor plasmid (pEAF), respectively. EPEC colonizes the proximal small intestine, where it adheres to epithelial cells forming microcolonies. Microcolony formation is one of the initial steps in biofilm development, but, very little is known regarding biofilm formation by EPEC strains. Our laboratory investigated the ability of biofilm formation in a collection of 223 EPEC strains (70 typical and 153 atypical) belonged to EPEC and non-EPEC serogroups. Biofilm formation occurred in 11/ tEPEC and 37 aEPEC strains from different serotypes. Typical EPEC formed biofilm on a polystyrene plate at 37C, whereas aEPEC strains formed at 26C in LB, or LBNS. The gene sequences related to type 1 fimbriae, curli, flagellin, antigen 43, and the autotransporter proteins of enterohemorrhagic E. coli EhaA, EhaB (ehaBa and ehaBb) and Cah were identified in most of the typical and atypical biofilm-producing strains. The objetive of this study was to continue the studies of our laboratory, identifying the genetic determinant involved in the biofilm formation of one tEPEC strain. We began the study by selecting the tEPEC strain between the group of the strains previously characterized as biofilm forming. Biofilm formation quantification results showed that three strains presented strong biofilm and seven strains presented moderate biofilm at 37ºC in LBNS medium. Among the 3 strains, T29 (O119: H6) strain was selected for presenting a robust biofilm on abiotic surface and a dense pellicle at the air-liquid interface. The T29 strain (O119: H6) was mutagenized with the transposon mini-Tn10, and four mutants (M6, M21, M160 and M175) were identified that lost the ability to form biofilm on polystyrene surface and pellicle at the liquid-air interface. The DNA flanked by the transposon insert was cloned into the plasmid vector pUC19, and the clones obtained were sequenced. BLASTN analysis of the 529 bp and 322 bp sequences obtained from the sequencing of clones M6.1 and M21.1, respectively, revealed homology with the enzyme aspartate ammonia lyase and fimD gene related to fimbriae type 1. Studies are in progress with mutants M160 and M175.