Análise molecular e fenotípica da virulência em estirpes de Eschericha coli isoladas de alimentos prontos-a-comer (RTE)

• Author(s): Guerreiro, Suse Cristina Alves
• Date: 2012
• Persistent ID: http://hdl.handle.net/10400.5/5357
• Origin: Repositório da UTL
• Subject(s): Escherichia coli; cytotoxicity; Vero cells; virulence factors; multiplex PCR; ready-to-eat food
• Document Type: Master thesis
• Language: Portuguese
• Advisor(s): Brito, Maria Luísa de Castro; Machado, Jorge Pinheiro da Costa
• Contributor(s): Repositório da Universidade de Lisboa

Description

Mestrado em Engenharia Alimentar - Qualidade e Segurança Alimentar - Instituto Superior de Agronomia

The bacteria Escherichia coli diarrheagenic (DEC) are an important cause of gastrointestinal diseases. The DEC are classified into: enteropathogenic, enterohaemorrhagic, enterotoxigenic, enteroaggregative, enteroinvasive and diffusely adherent, due to the presence of certain virulence factors. The toxins LT, ST, EAST1 and VT are encoded by the genes lt, st, astA and stx, respectively. The intimin, encoded by the gene eae, causes the A/E lesions (attaching and effacing), which favors the adherence of bacteria to the intestine, being also an important factor of pathogenicity. This study was aimed to evaluate the virulence of 36 isolates of E. coli from ready-to-eat (RTE) food, by investigating the presence of the stx, lt, st, astA and eae genes (Multiplex PCR) and by measuring the activity of the enzyme lactate dehydrogenase (LDH) from Vero cells and microscopic observation (phase contrast and DAPI) of these cells, exposed to bacterial toxins. Five isolates showed the stx gene, one of which also amplified the eae, four isolates, the lt gene and three, the st. Cytotoxicity tests and microscopic analysis of exposed Vero cells validate the results obtained by multiplex PCR, being that the isolates stx+ induced higher release (p ≤ 0.05) of LDH (19.9 a 32%) than the isolates stx- (4.6 a 9.1%), having the first ones caused extensive cytopathic effects.